Ice-cold Saline Research Articles

A suspected case of malignant hyperthermia that was successfully treated with dantrolene administration via nasogastric tube

A suspected case of malignant hyperthermia that was successfully treated with dantrolene administration via nasogastric tube

Anesthetic management of Prader–Willi syndrome: what if neuromuscular relaxants could not be avoided?

To the Editor: We read with interest the article by Meco et al. [1] concerning the anesthetic management of a 16-month-old child with the Prader–Willi syndrome avoiding the use of neuromuscular blockers. We would like to add our experience with anesthetic management involving the use of succinylcholine and non-depolarizing neuromuscular blockers in a child with the Prader–Willi syndrome because avoiding these drugs may not be possible in all situations. A 5-year-old male child with Prader–Willi syndrome was scheduled for laparoscopy and repair of bilateral undescended testes. Preanesthetic examination revealed obesity (weight 30 kg, height 96 cm, body mass index 32 kg/m), short stature, small hands and feet, cryptorchid testes, and hypoplastic dental enamel. Following an inhalation induction with sevoflurane 8% in 100% oxygen, we succeed in obtaining venous access only after multiple failed attempts. However, the procedure was complicated by severe laryngospasm with an episode of desaturation (SpO2 85%). Intravenous succinylcholine 50 mg was given immediately and effective bag-mask ventilation could then be delivered. The trachea was secured using a 4.5-mm cuffed endotracheal tube. The patient was then maintained on nitrous oxide–oxygen–sevoflurane and atracurium for relaxation. Analgesia was achieved with intravenous fentanyl 30 lg. His initial esophageal temperature was 36.5 C but this increased to 39.4 C after 40 min. This change was accompanied by an increase in heart rate and end-tidal carbon dioxide concentration. An arterial blood gas revealed respiratory acidosis. The patient was actively cooled with cold sponges, application of ice packs in the axilla, and gastric lavage using ice-cold saline. His temperature dropped to 37.8 C after 60 min. No dantrolene sodium was given. The total duration of surgery was 130 min. The neuromuscular blockade was then reversed using atropine and neostigmine, without difficulty. The patient was extubated awake with good respiratory efforts. There were no episodes of hyperthermia and obstructive sleep apnea in the postoperative period. The CPK obtained 8 h following anesthesia was 25 units per liter (normal, 8–132). Certainly, use of anesthesia techniques [1, 2] avoiding the use of neuromuscular blockade in the Prader–Willi syndrome may reduce the risk of postoperative complications associated with this syndrome. However, avoiding neuromuscular blockade altogether may not be possible in all situations. We report this anesthetic experience because we believe that as experience is gained, our understanding increases concerning anesthetizing children with the Prader–Willi syndrome. (1) A narcotic-relaxant technique can be utilized safely in children with the Prader–Willi syndrome [3]. Both succinylcholine and non-depolarizing relaxants can be safely used in patients with Prader–Willi syndrome [4]. (2) Although thermoregulatory dysfunctions including intraoperative hypothermia are common in patients with Prader–Willi syndrome, a relationship to malignant hyperthermia has not been established [5]. Thus, the intraoperative increase in temperature with the use of succinylcholine does not always indicate malignant hyperthermia in these patients. (4) Despite the presence of hypotonia, neuromuscular blockade with good reversal is possible [3]. A. Jain (&) I. Bala J. K. Makkar Department of Anesthesia and Intensive Care, Post Graduate Institute of Medical Education and Research, Chandigarh 160012, India e-mail: amitvasujain@gmail.com

Cold Saline Lavage for Removal of Incarcerated Porous Ingrowth Stems

A patient with a history of total knee arthroplasty and 5 subsequent revisions presented with pain. His preoperative construct consisted of a revision implant with porous in-growth stem extensions. He was indicated for revision surgery for a painful implant with progressive arthrofibrosis. The surgery proceeded uneventfully until we were unable to loosen the proximal portion of the ingrown femoral stem. After the distal femur was removed, a trephine was used to break the bone ingrowth from the distal portion of the stem. Flexible osteotomes were used to loosen the bone-prosthetic interface, but they were unable to penetrate deep enough, and they eventually bent under the applied loads. Nonflexible osteotomes were also used without success, and we were still unable to remove the stem from within the femur. Slap hammers, vice grips, and other devices were used to try to remove the stem, but attempts were unsuccessful. We hung a 3-liter bag of ice-cold (4 °C) saline and ran all 3 liters of saline, under gravity pressure, retrograde down the femoral shaft in an attempt to contract the metal and allow for the removal of the stem. We then attached a stem extension screw to the incarcerated femoral stem and were able to remove it with minimal effort using a back-slap device. We inspected the prosthesis and found no abnormalities of the stem to explain its incarceration and no iatrogenic perforations in the femur. This case demonstrates a useful technique that should be kept in a revision surgeon's armamentarium for the removal of incarcerated porous ingrowth stems.

Abstract 98: Initiation of Large-Volume, Ice-Cold Saline Infusion During Initial Resuscitation Attempts for Out-of-Hospital Cardiac Arrest Results in Near-Target Temperatures for Therapeutic Hypothermia Upon Emergency Department Arrival

Background: New York City Project Hypothermia is a collaborative effort involving the Fire Department of New York (FDNY), Greater New York Hospital Association, Health and Hospitals Corporation, the Regional Emergency Medical Advisory Committee, and the New York State Department of Health. As part of this effort, the FDNY implemented a pilot protocol in the New York City 9-1-1 System on August 1, 2010 that introduced the induction of therapeutic hypothermia during initial resuscitation efforts via large-volume, pressure infused, ice-cold saline administration. Purpose: We sought to assess the effects of the intra-arrest initiation of an infusion of up to 30cc/kg of ice-cold saline on core body temperature for out-of-hospital cardiac arrest (OOHCA). Methods: A convenience sample of out-of-hospital cardiac arrest patients for whom core temperature monitoring via esophageal temperature probes was available were analyzed. Results: There were a total of 78 patients for whom core temperature (esophageal) data was available both at the time hypothermia induction and upon ED arrival. Eight patients (10.3%) were reported to have developed clinical signs of pulmonary edema during the infusion and were excluded from this analysis due to the discontinuation of the large-volume infusion. Among the remaining 70 patients, an average of 1,361mls of ice-cold saline was administered prior to emergency department arrival. The average initial temperature for these patients at the time hypothermia induction was 35.1 o C (range 32.7-38.0 o C). The average temperature reduction as a result of the large-volume, ice-cold saline infusion was -0.92 o C, with an average core temperature upon emergency department arrival of 34.2 o C. Conclusion: Large volume, ice-cold saline infusion during initial resuscitation efforts allows patients to arrive in the emergency department with a core temperature near the target range for therapeutic hypothermia. Further study is needed to assess the effects of early induction of hypothermia on immediate and long-term survival.

Abstract 242: Incidence and Relevance of Pulmonary Edema as a Result of Large-Volume, Ice-Cold Saline Infusion for the Induction of Hypothermia During Out-of-Hospital Cardiopulmonary Resuscitation

Background: New York City Project Hypothermia is a collaborative effort involving the Fire Department of New York (FDNY), Greater New York Hospital Association, Health and Hospitals Corporation, the Regional Emergency Medical Advisory Committee, and the New York State Department of Health. As part of this effort, the FDNY implemented a pilot protocol that introduced the induction of therapeutic hypothermia during initial resuscitation efforts for nontraumatic adult cardiac arrests. Purpose: We sought to measure the incidence of pulmonary edema among patients receiving large-volume, ice-cold saline during initial resuscitation efforts and to assess the impact of this condition on immediate outcomes. Methods: Out-of-hospital cardiac arrest data was analyzed for the following periods: August 1, 2009 - May 31, 2010 (historical control group) and August 1, 2010 - May 31, 2011 (study group). Pulmonary edema was defined as having occurred if clinical signs and symptoms were reported by the on-scene paramedics during a standardized post-resuscitation interview. Data were analyzed using Fisher's exact test, and a p <0.05 was considered significant. Results: There were 5,582 resuscitations during the control period and 4,727 resuscitations in the study period. Return of spontaneous circulation (ROSC) and sustained ROSC occurred among 29.0% and 21.9% of the control group and 31.7% and 24.1% of the study group, respectively. A total of 361/4,727 patients (7.6%) in the study period were experienced pulmonary edema. The ROSC and sustained ROSC rates for this population were 37.1% and 25.2%, respectively. These outcomes were significantly improved as compared to both the control group and the remainder of the study group (all p <0.01). Conclusion: Clinical signs of pulmonary edema develop in some out-of-hospital cardiac arrest patients receiving large volume, ice-cold saline infusion for the purpose of initiating therapeutic hypothermia. Given the improved immediate outcomes among these patients, the clinical relevance of this clinical condition is of uncertain significance.

Abstract 97: Intra-arrest Induction of Therapeutic Hypothermia via Large-Volume, Ice-Cold Saline Infusion Results in Improved Outcomes Among Out-of-Hospital Cardiac Arrests of Cardiac Etiology

Background: New York City Project Hypothermia is a collaborative effort involving the Fire Department of New York (FDNY), Greater New York Hospital Association, Health and Hospitals Corporation, the Regional Emergency Medical Advisory Committee, and the New York State Department of Health. As part of this effort, the FDNY implemented a new pilot protocol in the New York City 9-1-1 System that introduced the induction of therapeutic hypothermia during initial resuscitation efforts via large-volume ice-cold saline infusion. Purpose: We sought to assess the effects of this protocol on immediate survival end-points following out-of-hospital cardiac arrest (OOHCA) of cardiac etiology. Methods: OOHCA data was analyzed for the following periods: August 1, 2009 - May 31, 2010 (historicalcontrol group) and August 1, 2010 - May 31, 2011 (study group). Except for the intra-arrest induction of hypothermia, no other aspect of the regional resuscitation protocols differed between the two periods. Standard Utstein definitions were utilized. Fisher's exact test was utilized, and a p <0.05 was considered significant. Results: 4,459 / 5,582 (79.9%) of resuscitations during the control period and 3,581 / 4,727 (75.8%) of resuscitations during the study period were of cardiac etiology. The groups did not differ with respect to age, gender, race, response time, bystander witnessed status, or frequency of bystander CPR. Patients in the study period were less likely to have an EMS-witnessed arrest (6.5% vs 7.8%, p=0.03). Return of spontaneous circulation (ROSC) and sustained ROSC were improved in the study group as compared to the control group: 31.1% vs 26.8% (p<0.0001) and 23.6% vs 19.9% (p<0.0001), respectively. Conclusion: The initiation of therapeutic hypothermia via large volume, ice-cold saline infusion improves immediate survival for non-traumatic out-of-hospital cardiac arrest of cardiac etiology. Further study is required to identify specific populations for whom this therapy is most appropriate.

Abstract 2: Intra-arrest Induction of Therapeutic Hypothermia via Large-Volume Ice-Cold Saline Infusion Improves Immediate Outcomes for Out-of-Hospital Cardiac Arrest

Background: New York City Project Hypothermia is a collaborative effort involving the Fire Department of New York (FDNY), Greater New York Hospital Association, Health and Hospitals Corporation, the Regional Emergency Medical Advisory Committee, and the New York State Department of Health. As part of this effort, the FDNY implemented a pilot protocol in the New York City 9-1-1 System on August 1, 2010 that introduced the induction of therapeutic hypothermia during initial resuscitation efforts via large-volume ice-cold saline infusion. Purpose: We sought to assess the effects of this protocol on immediate survival end-points following out-of-hospital cardiac arrest (OOHCA). Methods: OOHCA data was analyzed for the following periods: August 1, 2009 - May 31, 2010 (historicalcontrol group) and August 1, 2010 - May 31, 2011 (study group). Except for the intra-arrest induction of hypothermia, no other aspect of the regional resuscitation protocols differed between the two periods. Standard Utstein definitions were utilized. Due to the large sample sizes, Chi-square analyses without Yates' correction were utilized, and a p <0.05 was considered significant. Results: 5,582 resuscitations for nontraumatic adult cardiac arrests during the control period were compared to 4,727 resuscitations in the study period that included the intra-arrest induction of hypothermia. The groups did not differ with respect to age, response time, bystander witnessed status, or frequency of bystander CPR. Patients in the study period were less likely to be male (52.3% vs 54.6%, p = 0.019), less likely to be white (32.8% vs 35.1%, p = 0.013), and less likely to have an EMS-witnessed arrest (8.3% vs 9.5%, p=0.026). Return of spontaneous circulation (ROSC) and sustained ROSC were improved in the study group as compared to the control group: 31.7% vs 29.0% (p=0.003) and 24.1% vs 21.9% (p=0.0014), respectively. Conclusion: The initiation of therapeutic hypothermia via large volume, ice-cold saline infusion results in improved immediate survival for non-traumatic out-of-hospital cardiac arrest. To our knowledge, this is the first report to describe the use of intra-arrest hypothermia in humans and to demonstrate such an effect.

Role of tumor suppressor p53 in megakaryopoiesis and platelet function

The pathobiological role of p53 has been widely studied, however, its role in normophysiology is relatively unexplored. We previously showed that p53 knock-down increased ploidy in megakaryocytic cultures. This study aims to examine the effect of p53 loss on invivo megakaryopoiesis, platelet production, and function, and to investigate the basis for greater ploidy in p53(-/-) megakaryocytic cultures. Here, we used flow cytometry to analyze ploidy, DNA synthesis, and apoptosis in murine cultured and bone marrow megakaryocytes following thrombopoietin administration and to analyze fibrinogen binding to platelets invitro. Culture of p53(-/-) marrow cells for 6 days with thrombopoietin gave rise to 1.7-fold more megakaryocytes, 26.1% ± 3.6% of which reached ploidy classes ≥64 N compared to 8.2% ± 0.9% of p53(+/+) megakaryocytes. This was due to 30% greater DNA synthesis in p53(-/-) megakaryocytes and 31% greater apoptosis in p53(+/+) megakaryocytes by day 4 of culture. Although the bone marrow and spleen steady-state megakaryocytic content and ploidy were similar in p53(+/+) and p53(-/-) mice, thrombopoietin administration resulted in increased megakaryocytic polyploidization in p53(-/-) mice. Although their platelet counts were normal, p53(-/-) mice exhibited significantly longer bleeding times and p53(-/-) platelets were less sensitive than p53(+/+) platelets to agonist-induced fibrinogen binding and P-selectin secretion. In summary, our invivo and exvivo studies indicate that p53 loss leads to increased polyploidization during megakaryopoiesis. Our findings also suggest for the first time a direct link between p53 loss and the development of fully functional platelets resulting in hemostatic deficiencies.

Design of the RINSE Trial: The Rapid Infusion of cold Normal Saline by paramedics during CPR

BackgroundThe International Liaison Committee on Resuscitation (ILCOR) now recommends therapeutic hypothermia (TH) (33°C for 12-24 hours) as soon as possible for patients who remain comatose after resuscitation from shockable rhythm in out-of-hospital cardiac arrest and that it be considered for non shockable rhythms. The optimal timing of TH is still uncertain. Laboratory data have suggested that there is significantly decreased neurological injury if cooling is initiated during CPR. In addition, peri-arrest cooling may increase the rate of successful defibrillation. This study aims to determine whether paramedic cooling during CPR improves outcome compared standard treatment in patients who are being resuscitated from out-of-hospital cardiac arrest.Methods/DesignThis paper describes the methodology for a definitive multi-centre, randomised, controlled trial of paramedic cooling during CPR compared with standard treatment. Paramedic cooling during CPR will be achieved using a rapid infusion of large volume (20-40 mL/kg to a maximum of 2 litres) ice-cold (4°C) normal saline.The primary outcome measure is survival at hospital discharge. Secondary outcome measures are rates of return of spontaneous circulation, rate of survival to hospital admission, temperature on arrival at hospital, and 12 month quality of life of survivors.DiscussionThis trial will test the effect of the administration of ice cold saline during CPR on survival outcomes. If this simple treatment is found to improve outcomes, it will have generalisability to prehospital services globally.Trial RegistrationClinicalTrials.gov: NCT01172678

Antioxidation effect of Simvastatin in rabbit hippocampus: role of superoxide dismutase, glutathione peroxidase, malondialdehyde and haem oxygenase-1

ObjectivesHypercholesterolemia contributes to coronary artery disease progression but little is known about its effect on hippocampus. The pleiotropic effects of HMG-CoA inhibitors (statins), which include anti-inflammation, antioxidation and immunomodulation. It...

Role of the Transmembrane Domain 4/Extracellular Loop 2 Junction of the Human Gonadotropin-releasing Hormone Receptor in Ligand Binding and Receptor Conformational Selection

Recent crystal structures of G protein-coupled receptors (GPCRs) show the remarkable structural diversity of extracellular loop 2 (ECL2), implying its potential role in ligand binding and ligand-induced receptor conformational selectivity. Here we have applied molecular modeling and mutagenesis studies to the TM4/ECL2 junction (residues Pro(174(4.59))-Met(180(4.66))) of the human gonadotropin-releasing hormone (GnRH) receptor, which uniquely has one functional type of receptor but two endogenous ligands in humans. We suggest that the above residues assume an α-helical extension of TM4 in which the side chains of Gln(174(4.60)) and Phe(178(4.64)) face toward the central ligand binding pocket to make H-bond and aromatic contacts with pGlu(1) and Trp(3) of both GnRH I and GnRH II, respectively. The interaction between the side chains of Phe(178(4.64)) of the receptor and Trp(3) of the GnRHs was supported by reciprocal mutations of the interacting residues. Interestingly, alanine mutations of Leu(175(4.61)), Ile(177(4.63)), and Met(180(4.66)) decreased mutant receptor affinity for GnRH I but, in contrast, increased affinity for GnRH II. This suggests that these residues make intramolecular or intermolecular contacts with residues of transmembrane (TM) domain 3, TM5, or the phospholipid bilayer, which couple the ligand structure to specific receptor conformational switches. The marked decrease in signaling efficacy of I177A and F178A also indicates that IIe(177(4.63)) and Phe(178(4.64)) are important in stabilizing receptor-active conformations. These findings suggest that the TM4/ECL2 junction is crucial for peptide ligand binding and, consequently, for ligand-induced receptor conformational selection.

Cooperative Role of Nectin-Nectin and Nectin-Afadin Interactions in Formation of Nectin-based Cell-Cell Adhesion

The nectin cell adhesion molecules interact in trans with each other through their extracellular regions and with afadin through their cytoplasmic tails, forming adherens junctions in cooperation with cadherins. In a single cell, Necl-5 (nectin-like molecule-5) localizes at the leading edge and regulates directional cell movement in response to a chemoattractant. In such a single cell, afadin also localizes at the leading edge without interacting with nectins or Necl-5. It remains unknown how the nectin-nectin and nectin-afadin interactions are initiated when moving cells contact each other to initiate the formation of adherens junctions. We show here that the Necl-5-nectin interaction induced by cell-cell contact enhances the nectin-afadin interaction. This interaction then enhances the nectin-nectin interaction, which further enhances the nectin-afadin interaction in a positive feedback manner. Thus, the Necl-5-nectin, nectin-nectin, and nectin-afadin interactions cooperatively increase the clustering of the nectin-afadin complex at the cell-cell contact sites, promoting the formation of the nectin-based cell-cell adhesion.

MiRNA Expression Profile after Status Epilepticus and Hippocampal Neuroprotection by Targeting miR-132

When an otherwise harmful insult to the brain is preceded by a brief, noninjurious stimulus, the brain becomes tolerant, and the resulting damage is reduced. Epileptic tolerance develops when brief seizures precede an episode of prolonged seizures (status epilepticus). MicroRNAs (miRNAs) are small, noncoding RNAs that function as post-transcriptional regulators of gene expression. We investigated how prior seizure preconditioning affects the miRNA response to status epilepticus evoked by intra-amygdalar kainic acid in mice. The miRNA was extracted from the ipsilateral CA3 subfield 24 hours after focal-onset status epilepticus in animals that had previously received either seizure preconditioning (tolerance) or no preconditioning (injury), and mature miRNA levels were measured using TaqMan low-density arrays. Expression of 21 miRNAs was increased, relative to control, after status epilepticus alone, and expression of 12 miRNAs was decreased. Increased miR-132 levels were matched with increased binding to Argonaute-2, a constituent of the RNA-induced silencing complex. In tolerant animals, expression responses of >40% of the injury-group-detected miRNAs differed, being either unchanged relative to control or down-regulated, and this included miR-132. In vivo microinjection of locked nucleic acid-modified oligonucleotides (antagomirs) against miR-132 depleted hippocampal miR-132 levels and reduced seizure-induced neuronal death. Thus, our data strongly suggest that miRNAs are important regulators of seizure-induced neuronal death.

Involvement of Protein Kinase Cδ/Extracellular Signal-regulated Kinase/Poly(ADP-ribose) Polymerase-1 (PARP-1) Signaling Pathway in Histamine-induced Up-regulation of Histamine H1 Receptor Gene Expression in HeLa Cells

The histamine H(1) receptor (H1R) gene is up-regulated in patients with allergic rhinitis. However, the mechanism and reason underlying this up-regulation are still unknown. Recently, we reported that the H1R expression level is strongly correlated with the severity of allergic symptoms. Therefore, understanding the mechanism of this up-regulation will help to develop new anti-allergic drugs targeted for H1R gene expression. Here we studied the molecular mechanism of H1R up-regulation in HeLa cells that express H1R endogenously in response to histamine and phorbol 12-myristate 13-acetate (PMA). In HeLa cells, histamine stimulation caused up-regulation of H1R gene expression. Rottlerin, a PKCδ-selective inhibitor, inhibited up-regulation of H1R gene expression, but Go6976, an inhibitor of Ca(2+)-dependent PKCs, did not. Histamine or PMA stimulation resulted in PKCδ phosphorylation at Tyr(311) and Thr(505). Activation of PKCδ by H(2)O(2) resulted in H1R mRNA up-regulation. Overexpression of PKCδ enhanced up-regulation of H1R gene expression, and knockdown of the PKCδ gene suppressed this up-regulation. Histamine or PMA caused translocation PKCδ from the cytosol to the Golgi. U0126, an MEK inhibitor, and DPQ, a poly(ADP-ribose) polymerase-1 inhibitor, suppressed PMA-induced up-regulation of H1R gene expression. These results were confirmed by a luciferase assay using the H1R promoter. Phosphorylation of ERK and Raf-1 in response to PMA was also observed. However, real-time PCR analysis showed no inhibition of H1R mRNA up-regulation by a Raf-1 inhibitor. These results suggest the involvement of the PKCδ/ERK/poly(ADP-ribose) polymerase-1 signaling pathway in histamine- or PMA-induced up-regulation of H1R gene expression in HeLa cells.

Cardiac output can be measured with the transpulmonary thermodilution method in a paediatric animal model with a left-to-right shunt

The transpulmonary thermodilution (TPTD) technique for measuring cardiac output (CO) has never been validated in the presence of a left-to-right shunt. In this experimental, paediatric animal model, nine lambs with a surgically constructed aorta-pulmonary left-to-right shunt were studied under various haemodynamic conditions. CO was measured with closed and open shunt using the TPTD technique (CO(TPTD)) with central venous injections of ice-cold saline. An ultrasound transit time perivascular flow probe around the main pulmonary artery served as the standard reference measurement (CO(MPA)). Seven lambs were eligible for further analysis. Mean (sd) weight was 6.6 (1.6) kg. The mean CO(MPA) was 1.21 litre min(-1) (range 0.61-2.06 l min(-1)) with closed shunt and 0.93 litre min(-1) (range 0.48-1.45 litre min(-1)) with open shunt. The open shunt resulted in a mean Q(p)/Q(s) ratio of 1.8 (range 1.6-2.4). The bias between the two CO methods was 0.17 litre min(-1) [limits of agreement (LOA) of 0.27 litre min(-1)] with closed shunt and 0.14 litre min(-1) (LOA of 0.32 litre min(-1)) with open shunt. The percentage errors were 22% with closed shunt and 34% with open shunt. The correlation (r) between the two methods was 0.93 (P<0.001) with closed shunt and 0.86 (P<0.001) with open shunt. The correlation (r) between the two methods in tracking changes in CO (ΔCO) during the whole experiment was 0.94 (P<0.0001). The TPTD technique is a feasible method of measuring CO in paediatric animals with a left-to-right shunt.

Identification of a Negative Regulatory Region for the Exchange Activity and Characterization of T332I Mutant of Rho Guanine Nucleotide Exchange Factor 10 (ARHGEF10)

The T332I mutation in Rho guanine nucleotide exchange factor 10 (ARHGEF10) was previously found in persons with slowed nerve conduction velocities and thin myelination of peripheral nerves. However, the molecular and cellular basis of the T332I mutant is not understood. Here, we show that ARHGEF10 has a negative regulatory region in the N terminus, in which residue 332 is located, and the T332I mutant is constitutively active. An N-terminal truncated ARHGEF10 mutant, ARHGEF10 ΔN (lacking amino acids 1-332), induced cell contraction that was inhibited by a Rho kinase inhibitor Y27632 and had higher GEF activity for RhoA than the wild type. The T332I mutant also showed the phenotype similar to the N-terminal truncated mutant. These data suggest that the ARHGEF10 T332I mutation-associated phenotype observed in the peripheral nerves is due to activated GEF activity of the ARHGEF10 T332I mutant.

Differential Impact of Caveolae and Caveolin-1 Scaffolds on The Membrane Raft Proteome

Caveolae, a class of cholesterol-rich lipid rafts, are smooth invaginations of the plasma membrane whose formation in nonmuscle cells requires caveolin-1 (Cav1). The recent demonstration that Cav1-associated cavin proteins, in particular PTRF/cavin-1, are also required for caveolae formation supports a functional role for Cav1 independently of caveolae. In tumor cells deficient for Golgi β-1,6N-acetylglucosaminyltransferase V (Mgat5), reduced Cav1 expression is associated not with caveolae but with oligomerized Cav1 domains, or scaffolds, that functionally regulate receptor signaling and raft-dependent endocytosis. Using subdiffraction-limit microscopy, we show that Cav1 scaffolds are homogenous subdiffraction-limit sized structures whose size distribution differs from that of Cav1 in caveolae expressing cells. These cell lines displaying differing Cav1/caveolae phenotypes are effective tools for probing the structure and composition of caveolae. Using stable isotope labeling by amino acids in cell culture, we are able to quantitatively distinguish the composition of caveolae from the background of detergent-resistant membrane proteins and show that the presence of caveolae enriches the protein composition of detergent-resistant membrane, including the recruitment of multiple heterotrimeric G-protein subunits. These data were further supported by analysis of immuno-isolated Cav1 domains and of methyl-β-cyclodextrin-disrupted detergent-resistant membrane. Our data show that loss of caveolae results in a dramatic change to the membrane raft proteome and that this change is independent of Cav1 expression. The proteomics data, in combination with subdiffraction-limit microscopy, indicates that noncaveolar Cav1 domains, or scaffolds are structurally and functionally distinct from caveolae and differentially impact on the molecular composition of lipid rafts.

Ex-VivoUreteroscopy at the Time of Live Donor Nephrectomy

Potential transplant renal allograft recipients exceed the number of donors. Our institution now considers patients with small, unilateral, nonobstructing, incidental renal calculi for possible renal donation. We adopted ex-vivo ureteroscopy (ExURS) to render these kidneys stone free at the time of renal transplantation. We examined the safety and efficacy of ExURS. After confirming a lack of significant metabolic defects on 24-hour urinalysis, 23 patients with small nonobstructing unilateral nephrolithiasis detected on preoperative CT angiography underwent donor nephrectomy. Immediately after cold perfusion, ExURS was performed with ice cold saline irrigation. Retrospective review was performed. Pyeloscopy was successfully performed in all 23 patients. A total of 28 calculi, mean largest diameter 3.9 mm (range 3-6 mm), were visualized in 19 kidneys. Basket extraction and holmium laser lithotripsy was performed in 12 and 6 kidneys, respectively. Treatment rendered 17/19 stone-containing kidneys stone free with a mean treatment time of 6.2 minutes (3-10 min). There were no intraoperative complications. Median serum creatinine level of recipients at 1 month and 1 year were 1.4 ± 1.8 mg/dL and 1.3 ± 0.6 mg/dL, respectively. At a median follow-up of 63 ± 47.2 months, there were no transplant urinary calculi among the recipients. ExURS safely renders live donor kidney allografts stone free with low risk of recurrence. When used appropriately, ExURS could safely increase the number of potential kidney donors and minimize the risk of adverse stone events.

Switching of N-Methyl-d-aspartate (NMDA) Receptor-favorite Intracellular Signal Pathways from ERK1/2 Protein to p38 Mitogen-activated Protein Kinase Leads to Developmental Changes in NMDA Neurotoxicity

Excitotoxicity mediated by overactivation of N-methyl-D-aspartate receptors (NMDARs) has been implicated in a variety of neuropathological conditions in the central nervous system (CNS). It has been suggested that N-methyl-D-aspartate (NMDA) neurotoxicity is developmentally regulated, but the definite pattern of the regulation has been controversial, and the underlying mechanism remains largely unknown. Here, we show that NMDA treatment leads to significant cell death in mature (9 and 12 days in vitro) hippocampal neurons or hippocampi of young postnatal day 12 and adult rats but not in immature (3 and 6 days in vitro) neurons or embryonic day 18 and neonatal rat hippocampi. In contrast, NMDA promotes survival of immature neurons against tropic deprivation. Interestingly, it is found that NMDA preferentially activates p38 MAPK in mature neuron and adult rat hippocampus, but it favors ERK1/2 activation in immature neuron and postnatal day 0 rat hippocampus. Moreover, it is shown that NMDA neurotoxicity in mature neuron is mediated via p38 MAPK activation, and neuroprotection in immature neuron is mediated via ERK1/2 activation, whereas all these effects are NR2B-containing NMDAR-dependent, as well as Ca(2+)-dependent. We also revealed that mature and immature neurons showed no difference in the amplitude of NMDA-induced intracellular calcium ([Ca(2+)](i)) increase. However, the basal level of [Ca(2+)](i) is shown to elevate with the maturation of neuron, and this elevation is attributable to the changes in NMDA neurotoxicity but not to the switch of the NMDAR signaling pathway. Taken together, our results suggest that a switch of NMDA receptor-favorite intracellular signal pathways from ERK1/2 to p38 MAPK and the elevated basal level of [Ca(2+)](i) with age might be critical for the developmental changes in NMDA neurotoxicity in the hippocampal neuron.

HSV-1 Cgal+ Infection Promotes Quaking RNA Binding Protein Production and Induces Nuclear-Cytoplasmic Shuttling of Quaking I-5 Isoform in Human Hepatoma Cells

Herpesvirus type 1 (HSV-1) based oncolytic vectors arise as a promising therapeutic alternative for neoplastic diseases including hepatocellular carcinoma. However, the mechanisms mediating the host cell response to such treatments are not completely known. It is well established that HSV-1 infection induces functional and structural alterations in the nucleus of the host cell. In the present work, we have used gel-based and shotgun proteomic strategies to elucidate the signaling pathways impaired in the nucleus of human hepatoma cells (Huh7) upon HSV-1 Cgal(+) infection. Both approaches allowed the identification of differential proteins suggesting impairment of cell functions involved in many aspects of host-virus interaction such as transcription regulation, mRNA processing, and mRNA splicing. Based on our proteomic data and additional functional studies, cellular protein quaking content (QKI) increases 4 hours postinfection (hpi), when viral immediate-early genes such as ICP4 and ICP27 could be also detected. Depletion of QKI expression by small interfering RNA results in reduction of viral immediate-early protein levels, subsequent decrease in early and late viral protein content, and a reduction in the viral yield indicating that QKI directly interferes with viral replication. In particular, HSV-1 Cgal(+) induces a transient increase in quaking I-5 isoform (QKI-5) levels, in parallel with an enhancement of p27(Kip1) protein content. Moreover, immunofluorescence microscopy showed an early nuclear redistribution of QKI-5, shuttling from the nucleus to the cytosol and colocalizing with nectin-1 in cell to cell contact regions at 16-24 hpi. This evidence sheds new light on mechanisms mediating hepatoma cell response to HSV-1 vectors highlighting QKI as a central molecular mediator.