Middle-Down Fragmentation for the Identification and Quantitation of Site-Specific Methionine Oxidation in an IgG1 Molecule

A middle-down LC/MS approach, for the rapid quantitation and characterization of site-specific methionine oxidation in a recombinant monoclonal IgG1 molecule, is described. An IgG1 antibody was digested with endoprotease LysC under limited proteolytic conditions to produce two major components; an antigen binding fragment (Fab) and a crystallizable fraction (Fc). These fractions were then reduced to produce three major species; light chain (LC), Fc/2 which is the C terminal region of the heavy chain (HC) and the N-terminal heavy chain region (Fd). These three fragments were separated by reversed-phase HPLC using a diphenyl column. The diphenyl column resolved site-specific methionine oxidation in all three subunits. Middle- down N-terminal sequencing with a LCT premier mass spectrometer was used to identify the sites of oxidation in the LC. Sites of oxidation in the Fc/2 were identified using middle-down collision-induced dissociation (CID) on a Qtof premier. This method allowed for the rapid quantitation and identification of oxidation on each methionine residue in an IgG1 molecule.     

Three balanced anesthetic techniques for neuroanesthesia: infusion of thiopental sodium with sufentanil or fentanyl compared with inhalation of isoflurane.

STUDY OBJECTIVE: To compare emergence from anesthesia and the hemodynamic and respiratory depressant effects of thiopental sodium infusion plus sufentanil or fentanyl with those of isoflurane as the primary component of a balanced technique for neuroanesthesia. DESIGN: Randomized, double-blind, prospective study. SETTING: University hospital and its affiliated Veterans Affairs Medical Center. PATIENTS: Thirty patients undergoing elective craniotomy for aneurysm or tumor. INTERVENTIONS: Thiopental with infusion of sufentanil 0.1 microgram/kg/hr, thiopental with infusion of fentanyl 1 microgram/kg/hr, or inhalation of 0.25% to 2% isoflurane as the major component of a balanced anesthesia technique that included nitrous oxide (N2O) and vecuronium (potency ratio of sufentanil to fentanyl, 10:1). MEASUREMENTS AND MAIN RESULTS: Intraoperative stress response (as indicated by intraoperative hypertension) was said to be the percentage of time the patient required administration of an antihypertensive drug, measuring from the first dose of thiopental to discontinuation of N2O at the end of the procedure, excluding any period of induced hypotension. Rapidity of emergence was measured by the number of minutes from discontinuation of N2O to first opening of the eyes on command. Adequacy of spontaneous ventilation was evaluated by determining partial pressure of arterial carbon dioxide 1, 2, and 3 hours after discontinuation of N2O. Extent of vasoactive drug administration for control of intraoperative hypertension (as determined by the clinicians caring for the patients) was described by minutes of vasodilator infusion and milligrams of propranolol or labetalol administered. The frequency of postoperative hypertension was defined as the number of patients in each group who required medication for postoperative hypertension. No significant differences in variables were found for thiopental/sufentanil, thiopental/fentanyl, or isoflurane when these drugs were used with N2O and vecuronium. CONCLUSIONS: Any one of these balanced anesthetic techniques appears appropriate for craniotomy.     

Plasma histamine and catecholamine levels during hypotension induced by morphine and compound 48/80

Histamine receptors are present in adrenergic terminals, and histamine is reported to inhibit release of the neurotransmitter norepinephrine (NE) at certain neuroeffector junctions. However, a physiological role for histamine in modifying adrenergic neurotransmission has not been established. To examine the interaction of elevated plasma histamine and catecholamine release, two compounds that release histamine, morphine (3 mg/kg), and compound 48/80 (0.5 mg/kg), were administered intravenously (i.v.). Plasma norepinephrine (NE) levels were used to monitor sympathetic nervous system activity, and plasma epinephrine (Epi) levels were used to monitor adrenal activity. Both morphine and compound 48/80 caused an immediate and marked increase in plasma histamine. Simultaneous with this increase, a marked decrease in mean arterial pressure occurred. Plasma NE levels increased in animals administered compound 48/80, but in morphine-treated animals, plasma NE levels did not change from pretreatment values. Plasma Epi levels increased in both groups, but the magnitude and duration of the responses differed. The results indicate that elevated plasma catecholamines can increase in response to histamine-induced hypotension but this effect can be suppressed by the central actions of morphine.     

Mast cell tryptase may modulate endothelial cell phenotype in healing myocardial infarcts

Mast cells and macrophages infiltrate healing myocardial infarcts and may play an important role in regulating fibrous tissue deposition and extracellular matrix remodelling. This study examined the time-course of macrophage and mast cell accumulation in healing infarcts and studied the histological characteristics and protease expression profile of mast cells in a canine model of experimental infarction. Although macrophages were more numerous than mast cells in infarct granulation tissue, macrophage density decreased during maturation of the scar, whereas mast cell numbers remained persistently elevated. During the inflammatory phase of infarction, newly recruited leucocytes infiltrated the injured myocardium and appeared to be clustered in close proximity to degranulating cardiac mast cells. During the proliferative phase of healing, mast cells had decreased granular content and were localized close to infarct neovessels. In contrast, macrophages showed no selective localization. Mast cells in healing canine infarcts were alcian blue/safranin-positive cells that expressed both tryptase and chymase. In order to explain the pro-inflammatory and angiogenic actions of tryptase--the major secretory protein of mast cells--its effects on endothelial chemokine expression were examined. Chemokines are chemotactic cytokines that play an important role in leucocyte trafficking and angiogenesis and are highly induced in infarcts. Tryptase, a proteinase-activated receptor (PAR)-2 agonist, induced endothelial expression of the angiogenic chemokines CCL2/MCP-1 and CXCL8/IL-8, but not the angiostatic chemokine CXCL10/IP-10. Endothelial PAR-2 stimulation with the agonist peptide SLIGKV induced a similar chemokine expression profile. Mast cell tryptase may exert its angiogenic effects in part through selective stimulation of angiogenic chemokines.     

Intracranial cavernoma. Surgical results of 47 cases

We report the surgical results in a series of 47 patients with cerebral cavernous malformation who had undergone surgery between 1973 and 1994, with a follow up ranging from 12 months to 24 years (mean: 4 years). They were divided in there groups according to their initial clinical presentation: epilepsy (31 cases), hemorrhage (11 cases) and neurological deficit (5 cases). Surgery consisted of cavernoma resection only (11 cases) or its extension to surrounding gliotic tissue (36 cases). Results are satisfactory: no surgical mortality, low morbidity (4 cases), no recurrent hemorrhage, seizures disappearance with anticonvulsant therapy stop (4 cases) or alleviation (20 cases). Only one patient died far from surgery (6 months) consequently to his initial bleeding, while all the others lead a normal active life. The therapeutic management, compared to the literature, pleads in favour of intentionally surgical attitude and gliotic tissue removal as often as reasonably possible.     

Monitoring of somatosensory evoked potentials during surgery for middle cerebral artery aneurysms

Somatosensory evoked potentials (SEPs) were monitored during 53 procedures for aneurysms of the middle cerebral artery (MCA). "Significant" changes were reported to the surgeon, who took corrective action when possible. Changes in the SEPs were categorized as follows: Type I, no change; Type II, significant change with complete return to baseline; Type III, significant change with incomplete return to baseline; Type IV, complete loss with no return; and Type V, no response at baseline. Only 1 of 37 patients with a Type I SEP had a new neurological deficit, and this was a patient who could not be examined for several days after surgery because he was in a pentobarbital coma. All 4 patients with Type III and IV changes had new postoperative neurological deficits. Perhaps of greater importance, 4 of 5 patients with Type II changes had no new deficit. These patients all had changes in SEPs that were completely reversible by clip adjustment (2), prompt removal of temporary clips (1), and inducing hypertension after aneurysm trapping (1). These cases may, therefore, represent instances in which SEP monitoring allowed the clinicians to prevent a neurological deficit. The MCA supplies the area of the somatosensory cortex that controls the hand. Median nerve SEPs are, therefore, a theoretically ideal monitor during surgery for MCA aneurysms. This study suggests that the results of MCA aneurysm surgery may be accurately predicted and improved with SEP monitoring.     

Diagnosis and Management of Atypical Hemolytic Uremic Syndrome In Children: Single Centre Experience

Atypical hemolytic uremic syndrome (aHUS) although rare is the commonest cause of acute renal failure (ARF) in children and has poor prognosis. We present single centre experience of aHUS. Thirty six children (29 males, 7 females) with mean age, 7.9 years presented with ARF, 2 children also had tonic–clonic type convulsions. Their hematology examination revealed hemolytic anemia with s. creatinine (SCr), 5.54 mg/dl. Acute HUS was observed in 75 %, acute on chronic HUS in 19.4 % and patchy cortical necrosis (PCN) in 5.6 % biopsies. Mean 5.4 plasma exchanges (PE) were carried out. Supportive management of anti-hypertensives and prednisone was also given. Recovery end points were establishment of urine output, improvement of SCr and hematological profile. Hematology and renal function profile improved variably in all children, 5.6 % died, relapse was observed in 80.5 % over mean 70 days; 13.9 % children are doing well over mean follow-up of 268.8 days. Thus poor prognosis was observed in 86.1 % children. Children with acute on chronic HUS and PCN did not recover. Six children who recovered had acute HUS. aHUS in Indian children occurs at an older age of around 8 years and chronic/irreversible changes on histopathology examination are harbingers of poor prognosis. PE is life-saving however further research for developing strategies to improve long-term survival is needed.