Modulation of p27(Kip1) levels by the cyclin encoded by Kaposi's sarcoma-associated herpesvirus

DNA tumour viruses have evolved a number of mechanisms by which they deregulate normal cellular growth control. We have recently described the properties of a cyclin encoded by human herpesvirus 8 (also known as Kaposi's sarcoma-associated herpesvirus) which is able to resist the actions of p16(Ink4a), p21(Cip1) and p27(Kip1) cdk inhibitors. Here we investigate the mechanism involved in the subversion of a G1 blockade imposed by overexpression of p27(Kip1). We demonstrate that binding of K cyclin to cdk6 expands the substrate repertoire of this cdk to include a number of substrates phosphorylated by cyclin-cdk2 complexes but not cyclin D1-cdk6. Included amongst these substrates is p27(Kip1) which is phosphorylated on Thr187. Expression of K cyclin in mammalian cells leads to p27(Kip1) downregulation, this being consistent with previous studies indicating that phosphorylation of p27(Kip1) on Thr187 triggers its downregulation. K cyclin expression is not able to prevent a G1 arrest imposed by p27(Kip1) in which Thr187 is mutated to non-phosphorylatable Ala. These results imply that K cyclin is able to bypass a p27(Kip1)-imposed G1 arrest by facilitating phosphorylation and downregulation of p27(Kip1) to enable activation of endogenous cyclin-cdk2 complexes. The extension of the substrate repertoire of cdk6 by K cyclin is likely to contribute to the deregulation of cellular growth by this herpesvirus-encoded cyclin.     

Catalytic subunit of DNA-dependent protein kinase: impact on lymphocyte development and tumorigenesis

The DNA-dependent protein kinase (DNA-PK) consists of a heterodimer DNA-binding complex, Ku70 and Ku80, and a large catalytic subunit, DNA-PKcs. To examine the role of DNA-PKcs in lymphocyte development, radiation sensitivity, and tumorigenesis, we disrupted the mouse DNA-PKcs by homologous recombination. DNA-PKcs-null mice exhibit neither growth retardation nor a high frequency of T cell lymphoma development, but show severe immunodeficiency and radiation hypersensitivity. In contrast to the Ku70-/- and Ku80-/- phenotype, DNA-PKcs-null mice are blocked for V(D)J coding but not for signal-end joint formation. Furthermore, inactivation of DNA-PKcs leads to hyperplasia and dysplasia of the intestinal mucosa and production of aberrant crypt foci, suggesting a novel role of DNA-PKcs in tumor suppression.     

Double mutant cycle analysis of aspartate 69, 97, and 103 to asparagine mutants in the m2 muscarinic acetylcholine receptor

Double mutant cycles provide a method for analyzing the effects of a mutation at a defined position in the protein structure on the properties of an amino acid at a second site. This approach was used to map potential interactions between aspartates 69, 97, and 103 in the m2 muscarinic acetylcholine receptor transmembrane helices 2 and 3. Receptors containing single and double aspartate to asparagine mutants were expressed in Chinese hamster ovary cells and their effects on ligand binding, signal transduction, and thermal stability determined. Analysis of the double mutant cycles showed that the mutations had approximately additive effects on ligand binding, signal transduction, and thermal stability. Ligand binding and thermal inactivation results support the conclusion that aspartate-103 is the ligand amine counterion. Effector coupling properties of the mutant receptors showed that aspartate-103 was also required for signal transduction activity. The mutation of aspartate-69 to asparagine completely eliminated signal transduction by the agonists acetylcholine, carbachol, and pilocarpine but not oxotremorine M, which caused reduced but significant inhibition of adenylyl cyclase and stimulation of phospholipase C. In contrast, adenylyl cyclase stimulation by the asparagine-69 mutant was elicited only by acetylcholine and carbachol but not by oxotremorine M. The variation in agonist-dependent effector coupling properties provides evidence that the asparagine-69 mutant can exist in activated receptor states that are different from the wild-type m2 muscarinic receptor.     

Voltage and pH-induced channel closure of porin OmpF visualized by atomic force microscopy

Gram-negative bacteria are protected by an outer membrane in which trimeric channels, the porins, facilitate the passage of small solutes. The pores are formed by membrane-spanning antiparallel beta-strands, which are connected by short turns on the periplasmic side and long loops on the extracellular side. Voltage and pH-dependent conformational changes of these extracellular loops have now been visualized by atomic force microscopy of two-dimensional crystals of Escherichia coli porin OmpF. The observed conformational changes accompany the closure of the channel entrance, and suggest that this is a mechanism that the cells have evolved to protect themselves from drastic changes of the environment.     

Anesthesia for patients with carcinoid syndrome

Carcinoid syndrome, although rare, can create serious problems to the anesthetist, both by the nature and variability of clinical manifestations and by the complications that can occur peroperatively. Recent research has led to a better understanding of the pathophysiology of the disease process. However, modern medicine is far from unraveling the precise nature and physiological effects of all the peptide mediators produced by these tumors. The severity of symptoms does not predict the severity of perioperative complications, so that patients with minor preoperative symptoms may have significant intraoperative complications. While urinary 5-HIAA levels provide a good indicator of disease progression, they cannot predict the degree or type of physiological response to intraoperative tumor manipulation. Indeed, urinary 5-HIAA may be normal both in the presence of a clinical diagnosis of carcinoid syndrome and in the face of a peroperative carcinoid crisis. The keys to successful anesthetic management of patients with carcinoid syndrome are good communication between endocrinologist, anesthetist, and surgeon and preoperative optimization of the patient. This includes appropriate investigation and treatment of the effects of carcinoid peptides and the prevention of their release from tumors. If possible, advice should be sought from centers with experience at managing this group of patients. Octreotide has largely replaced the use of other drugs both for symptomatic control and acute treatment of the symptoms associated with carcinoid syndrome. However, other drugs, such as aprotinin, still have a significant place in the symptomatic control and treatment of peroperative complications, as serotonin is only one of a large variety of peptides responsible for the clinical effects of this disease. Anesthetic technique should be aimed at minimizing carcinoid mediator release, in response to stress it induction of anesthesia and tracheal intubation and during tumor manipulation. It is equally important to prepare for carcinoid crisis by, for example, ordering drugs, which are otherwise uncommonly used in the theater setting, ahead of time. Cardiovascular instability, particularly hypotension, is common, so that full monitoring and vigilance is vital to predict its onset. The current surgical view of management is that, while curative resection of carcinoid tumors less than 2 cm in diameter with no evidence of invasion or metastatic spread is appropriate, patients with disseminated disease should be medically managed unless symptom control is poor. The exceptions to this are those patients with early and correctable carcinoid cardiac disease and those who require palliative procedures such as defunctioning obstructed bowel. Survival rates in patients following excision of gastric and appendical carcinoid tumors approach those of the general population as a whole and the chance of metastasis is extremely low. Only two series have been published in the anesthetic literature on anesthesia for patients with carcinoid syndrome, although there are many single-case reports. Despite the rarity of this syndrome, further formal studies into the anesthetic management of this condition should be encouraged.     

Systemic inflammatory response syndrome treatment by powdered sorbent pheresis: the BioLogic-Detoxification Plasma Filtration System

Systemic inflammatory response syndrome (SIRS) is one of the most common causes of death in intensive care unit patients. The detoxification plasma filtration (DTPF) system (HemoCleanse, Inc., West Lafayette, IN) combines the DT hemodiabsorption system in series with a push-pull pheresis PF system (a suspension of powdered sorbents surrounding 0.5 microm plasma filter membranes). Bidirectional plasma flow (at 80-100 ml/min) across the PF membranes provides direct contact between plasma proteins and powdered sorbents, as well as clearance of cytokines (tumor necrosis factor-alpha, interleukin-1beta, and interleukin-6) at a rate of 15-25 ml/min, without evidence of saturation for 90 minutes. In a U.S. Food and Drug Administration approved study we treated eight patients with SIRS and organ failure with a single DTPF treatment, using powdered charcoal as sorbent in four patients and powdered charcoal and silica in four patients. Treatments proceeded for 6 hours with proper heparin anticoagulation (activated clotting time 250-300 sec) and appeared safe. All patients improved during the treatments and each had increased blood pressure and decreased need for pressor agents. Plasma cytokine levels stabilized or decreased during treatment and were significantly lower the morning after treatment. Multiple organ dysfunction (MOD) and Acute Physiology Chronic Health Evaluation II scores and organ function gradually improved in most patients, and two patients survived for more than 28 days and two for more than 14 days. The DTPF System may prove beneficial in treatment of patients with sepsis.     

Delirium phenomenology illuminates pathophysiology, management, and course

The phenomenology of delirium has received little standardized longitudinal study but offers the prospect of valuable insights regarding clinical subtypes, differentiation from other neuropsychiatric disorders, identification of underlying pathophysiologies, management, and course. This review examines current approaches to the investigation of delirium phenomenology and how the findings to date illuminate our understanding of delirium. It concludes with recommendations for future investigations.