Detection of apoptosis in human and rat ovarian follicles

OBJECTIVE: The purpose of this study was to determine whether cells acquired from individual human preovulatory follicles undergo apoptosis (physiologic cell death) and, if so, to correlate the degree of apoptosis with characteristics of the follicles or the oocytes derived from the follicles. METHODS: We devised a sensitive nonradioactive method for detecting apoptotic DNA fragmentation in small numbers of cells derived from rat atretic follicles and follicular aspirates of patients undergoing assisted reproductive technologies. RESULTS: Using this method, apoptotic DNA was detected in rat atretic follicles, with optimal detection at 10-100 ng. Furthermore, apoptotic DNA was detected in some, but not all individual human follicular aspirates from several patients, and was found in follicles that produced oocytes that fertilized and developed into embryos. CONCLUSION: Apoptosis occurs in cells from human ovarian preovulatory follicles and may be a normal physiologic process of the follicle during luteinization.     

Cloning and assembly of PCR products using modified primers and DNA repair enzymes

We present a method for the creation of ligatable 3' overhangs by the incorporation of a modified base, uracil, at a specific position in the PCR primer and subsequent treatment with the DNA-modifying enzyme uracil DNA glycosylase and then either T4 endonuclease V or human apurinic/apyrimidinic endonuclease 1. In this study, we describe the cloning of a fragment specifying the chloramphenicol-resistance gene into a SacI vector site. To further test this method, three segments of the lacZ gene were amplified by PCR, and after treatment with the DNA-modifying enzymes, the properly oriented segments were ligated into a SacI-cleaved plasmid. Using the methods described, we were able to assemble PCR products into appropriate structures.     

Preoperative biliary drainage, colonisation of bile and postoperative complications in patients with tumours of the pancreatic head: a retrospective analysis of 241 consecutive patients

OBJECTIVE: To analyse the outcome of preoperative biliary drainage in patients being operated on for a tumour in the pancreatic head. DESIGN: Retrospective study. SETTING: University hospital, The Netherlands. SUBJECTS: Consecutive series of 241 patients. MAIN OUTCOME MEASURES: Decline in bilirubin concentrations and bacterial contamination of bile as a result of preoperative drainage. Incidence of postoperative complications in patients who underwent preoperative drainage and those who did not. RESULTS: 184/241 patients underwent preoperative biliary drainage. Endoscopic drainage was the most effective, shown by a median reduction in bilirubin concentrations of 82%, 74%, and 50% after endoscopic drainage (n = 149), papillotomy (n = 25) and external drainage (n = 10), respectively. Bacterial contamination of bile was significantly more common when an endoprosthesis was used, but did not result in a higher rate of infective complications. 163 Whipple's resections, 33 total pancreatectomies, and 45 biliary-enteric bypasses were performed. 137/241 (57%) patients had postoperative complications. There was no significant difference in the incidence of postoperative complications between patients who had preoperative biliary drainage and those who did not (p = 0.4).