Complete sequencing of the recombinant granulocyte-colony stimulating factor (filgrastim) and detection of biotinylation by mass spectrometry.

Granulocyte-colony stimulating factor stimulates production and antibacterial function of neutrophiles. Therapy using the recombinant protein drug represents a major step forward in oncology. The protein has not been, however, completely sequenced at the protein level and this formed the rationale of the current study. Recombinant G-CSF (filgrastim) was run on two-dimensional gel electrophoresis (2DE), the protein was in-gel digested with trypsin and chymotrypsin, and peptides were analysed on Nano-ESI-LC-MS/MS (high performance ion trap, HCT). Bioinformatic tools used were Mascot v2.2 and Modiro(TM) v1.1 softwares. A single spot was detected on 2DE and peptides resulting from in-gel digestion were unambiguously identified by the MS/MS approach leading to complete sequencing when both searching engines were applied. N-terminal methionine loss, N-terminal methionine oxidation and amidination were observed. Both softwares identified modifications. Complete sequencing by a non-sophisticated and rapid gel-based mass spectrometry approach confirmed the primary structure predicted from nucleic acid sequences. A chemical modification of glutamine 26 with the interim name PentylamineBiotin (Unimod accession number #800) compatible with biotinylation with 5-(biotinamido) pentylamine by the producer was detected by both softwares. Although there is some evidence that biotinylated G-CSF analogues are active, it remains open whether this modification may be responsible for the side effects observed or lead to changes of antigenicity.

Benthic foraminifera for heavy metal pollution monitoring: A case study from Burullus Lagoon of Egypt.

Sources of heavy metals pollution in the Burullus Lagoon include phosphate fertilizers, sewage and oil spills from fishing boats. The benthic species identified in this lagoon are Adelosina carinata striata (Wiesner), Quinqueloculina bosciana (d'Orbigny), Quinqueloculina seminulum (Linnaeus), Ammonia tepida (Cushman), Elphidium excavatum (Terquem). A. tepida is the most dominant species in the lagoon. It constitutes more than 97% of the total benthic foraminiferal assemblages reflecting tolerance to the very low salinity in the Burullus Lagoon. The intensity of deformation was severe exhibiting a peculiar change in the coiling direction in A. tepida with increase in cadmium concentration. A. tepida exhibited a great morphological variability and the recorded morphological abnormalities show high spire giving the spiroconvex test, additional chamber, aberrant chamber shape and size, twisted tests with elongated axes of rotation and complex deformities, whereas E. excavatum showed additional calcite secretion (tumors).

Primary angioplasty using a urokinase-coated hydrogel balloon in acute myocardial infarction during pregnancy.

A 38-year-old multigravid white female presented at 16 weeks gestation with an acute inferoposterolateral myocardial infarction. Emergent coronary angiography demonstrated a total proximal occlusion of a large dominant left circumflex artery with a filling defect at the site of the occlusion suggestive of thrombus. Primary angioplasty using a urokinase-coated hydrogel balloon resulted in successful recanalization of the vessel with restoration of normal TIMI Grade III flow and, most notably, apparent complete lysis of the intracoronary thrombus. After a subsequently uneventful pregnancy, a healthy baby was delivered.

Site-specific intracoronary thrombolysis with urokinase-coated hydrogel balloons: acute and follow-up studies in 95 patients.

Conventional balloon angioplasty in the presence of intracoronary thrombus is associated with an elevated risk for acute myocardial infarction, emergency bypass surgery, and death. The purpose of this study was to assess the safety and efficacy of a new technique to treat thrombus-containing stenoses consisting of the local delivery of urokinase directly to the site of intraluminal clot with hydrogel-coated balloons. Ninety-five patients with angiographically apparent intracoronary thrombus were treated with urokinase-coated hydrogel balloons either prior to (n = 74) or following (n = 21) conventional balloon angioplasty. Clinical diagnoses for the study group included acute myocardial infarction in 50 patients, postinfarction angina in 23 patients, and unstable angina in 22 patients. All hydrogel balloons were initially coated with urokinase by immersing the inflated balloon in a concentrated Abbokinase solution (50,000 units/ml) for 60 s. All patients were subsequently treated with drug-coated balloons using a balloon:artery ratio of 1:1, a mean of 2.2 +/- 1.2 inflations, and a mean total inflation time of 7.5 +/- 4.9 min. Use of urokinase-coated balloons resulted in angiographic disappearance of intracoronary thrombus in 78 patients, improvement in 14, and no change in the remaining 3 patients. Following hydrogel balloon use for the entire 95 patients, TIMI flow increased from 1.4 +/- 1.2 to 2.9 +/- 0.4, minimal lumen diameter increased from 0.4 +/- 0.4 to 2.0 +/- 0.6 mm, and thrombus score decreased from 2.0 +/- 0.9 to 0.2 +/- 0.6 (all P < 0.01). Procedural and early in-hospital complications were noted in 7 of the 95 patients (7.4%) and included abrupt closure in 3 patients, distal embolization in 1 patient, no reflow in 1 patient, sidebranch occlusion in 1 patient, and late closure in 1 patient. Two of the 3 patients with abrupt closure and the single patient with late closure required intracoronary stenting to maintain vessel patency. Two of these 7 patients sustained small myocardial infarctions, although no patient required emergency bypass surgery or experienced a procedural death. Late clinical follow-up (mean = 8.3 +/- 6.6 months; range = 2 wk to 29 mo) demonstrated adverse recurrent events in 29 of the 95 patients (30.5%), including death (n = 5), myocardial infarction (n = 2), and recurrence of angina (n = 22). The results of this study suggest that intracoronary thrombolysis can be safely and rapidly achieved by using limited quantities of urokinase delivered directly to the site of intraluminal clot with hydrogel balloons. Use of this technique may result in improved acute outcomes in comparison with conventional techniques currently being used to treat thrombus-containing stenoses.

Enhanced intracoronary thrombolysis with urokinase using a novel, local drug delivery system. In vitro, in vivo, and clinical studies.

BACKGROUND: Current pharmacological regimens for treating intracoronary thrombus in the cardiac catheterization laboratory generally involve the administration of thrombolytic agents that result in a systemic fibrinolytic state and/or require prolonged arterial drug infusion. The purpose of the present study was to assess a new technique for treating intracoronary thrombus consisting of the local infusion of limited quantities of urokinase with a novel drug delivery device. METHODS AND RESULTS: THe Dispatch coronary infusion catheter is a new local drug delivery system that allows for the prolonged infusion of therapeutic agents at an angioplasty site while distal coronary flow is maintained. Three experimental protocols were performed to determine the in vitro, in vivo, and clinical efficacy of this device. First, in vitro thrombolysis of fresh, porcine thrombus trapped in a 4-mm plastic tube with a 50% constriction and perfused with 20% porcine plasma was measured. Twenty-three thrombi were weighed before and after no treatment (n = 5), "systemic" urokinase administration (n = 4), local infusion of 150,000 U urokinase with a standard end-hole catheter (n = 4), local infusion of saline with the Dispatch catheter (n = 5), and local infusion of 150,000 U urokinase with the Dispatch catheter (n = 5). Second, 25 porcine coronary arteries in 23 pigs were dilated in vivo with conventional balloon angioplasty and then treated with 123I-labeled urokinase that was administered either by the Dispatch catheter (150,000 U; n = 16), intravenous systemic bolus (1,000,000 U; n = 3), guiding catheter infusion (500,000 U; n = 3), or local end-hole catheter infusion (150,000 U; n = 3). All vessels were subsequently harvested to quantify intramural deposition and subsequent washout of urokinase at the angioplasty site. Finally, 19 patients with angiographic evidence of intracoronary thrombus were treated with local urokinase infusion with the Dispatch catheter either before or after balloon angioplasty or directional atherectomy. In vitro studies demonstrated that infusion of urokinase with the Dispatch catheter decreased thrombus weight by 66% compared with no treatment (-25%), "systemic" urokinase administration (25%), end-hole catheter urokinase infusion (32%), or infusion of saline by the Dispatch catheter (32%) (P < or = .005). In vivo studies demonstrated immediate deposition of 0.12% of the urokinase delivered by the Dispatch catheter to the angioplasty site, compared with 0.0007% with systemic bolus, 0.003% with guiding catheter infusion, and 0.007% with local infusion with an end-hole catheter (P < .001). Urokinase deposited by the Dispatch catheter persisted intramurally for at least 5 hours. Patient studies demonstrated reduction of thrombus-containing stenoses and complete disappearance of intracoronary thrombus in all cases in which 150,000 U urokinase was locally infused over 30 minutes. There was no evidence of abrupt closure, distal embolization, or no reflow in any patient. CONCLUSIONS: Local urokinase delivery with the Dispatch catheter can result in rapid and complete intracoronary thrombolysis using substantially less drug than standard thrombolytic techniques. Intramural deposition of drug with this technique creates a local reservoir of urokinase that may provide prolonged thrombolytic activity at the infusion site.