ATP-mediated killing of intracellular mycobacteria by macrophages is a P2X(7)-dependent process inducing bacterial death by phagosome-lysosome fusion.

Mycobacterium tuberculosis survives within host macrophages by actively inhibiting phagosome fusion with lysosomes. Treatment of infected macrophages with ATP induces both cell apoptosis and rapid killing of intracellular mycobacteria. The following studies were undertaken to characterize the effector pathway(s) involved. Macrophages were obtained from p47(phox) and inducible NO synthase gene-disrupted mice (which are unable to produce reactive oxygen and nitrogen radicals, respectively) and P2X(7) gene-disrupted mice. RAW murine macrophages transfected with either the natural resistance-associated macrophage protein gene 1 (Nramp1)-resistant or Nramp1-susceptible gene were also used. The cells were infected with bacille Calmette-Guérin (BCG), and intracellular mycobacterial trafficking was analyzed using confocal and electron microscopy. P2X(7) receptor activation was essential for effective ATP-induced mycobacterial killing, as its bactericidal activity was radically diminished in P2X(7)(-/-) macrophages. ATP-mediated killing of BCG within p47(phox-/-), inducible NO synthase(-/-), and Nramp(s) cells was unaffected, demonstrating that none of these mechanisms have a role in the ATP/P2X(7) effector pathway. Following ATP stimulation, BCG-containing phagosomes rapidly coalesce and fuse with lysosomes. Blocking of macrophage phospholipase D activity with butan-1-ol blocked BCG killing, but not macrophage death. ATP stimulates phagosome-lysosome fusion with concomitant mycobacterial death via P2X(7) receptor activation. Macrophage death and mycobacterial killing induced by the ATP/P2X(7) signaling pathway can be uncoupled, and diverge proximal to phospholipase D activation.     

Maintenance,equipment and operational improvements within an established Fermentation Pilot Plant

The Merck Research Laboratories' Fermentation Pilot Plant was constructed and commissioned in the early 1980's. It is a highly instrumented and automated facility which serves to maximize data collection for a variety of experimental batches. To continue successful and versatile operation as the facility ages, various improvements in equipment, maintenance, instrumentation, cleaning and sterility have been implemented. The goals were to improve flexibility, minimize down time, increase measurement accuracy and reduce contamination to enhance facility output for process development and clinical material production.The authors acknowledge the assistance of Ralph Calderone, Dennis Marshall, Thomas Hartnett, the mechanics and supervisors of the maintenance and instrumentation department, as well as the various vendors involved in these studies.     

Teratogenic effects of trichloroacetonitrile in the Long-Evans rat

Trichloroacetonitrile (TCAN) is among a number of contaminants found in drinking water produced by reactions of chlorine with background organic material. Long-Evans rats were intubated with TCAN (0, 1, 7.5, 15, 35, 55 mg/kg) in a tricaprylin vehicle on gestation days 6-18. The highest dose tested (55 mg/kg) was lethal in 21% of the dams and produced 100% resorptions in two-thirds of the survivors. Only one maternal death was seen at the next-lower dose; however, fetal weight and viability were decreased in a dose-related manner. The percentage of embryolethality was 13.9% at the lowest dose and 78.4% at the high dose, with resorption of entire litters seen at 7.5 mg/kg and above. At all doses, cardiovascular (interventricular septal defect, levocardia, common carotid, and right-sided aortic arch and ductus arteriosus) and urogenital (hypoplastic, missing, misplaced and fused kidneys, and hypoplastic uterine horns) malformations were seen in the offspring. Frequency of these malformations was dose related, ranging from 8% to 35% at the 1.0- and 35-mg/kg doses, respectively. The incidence of total soft tissue malformations was statistically significant at 15 and 35 mg/kg. There were no significant treatment-related changes in the incidence of skeletal malformations. The no-effect dose was established by statistical analysis to be 1.0 mg/kg/day.     

Ernährungsphysiologische Untersuchungen an Lepidopteren

Zusammenfassung Die Untersuchungen wurden begonnen mit Darmsaft vonRaupen, und zwar von solchen Formen, deren Imagines Nektar aufnehmen (Pieris brassicae, napi undrapae, Deilephila euphorbiae, Sphinx ligustri) und solchen, deren Imagines verkümmerte Mundgliedmaßen besitzen und daher auf jede Nahrungsaufnahme verzichten (Dicranura vinula).Bei allen Formen konnte eine Invertase eine Diastase, eine Lipase und eine Protease festgestellt werden.Die Ernährung derImagines erwies sich von der der Raupen als grundverschieden. Dabei konnte ein tiefgreifender Unterschied zwischen saugenden und nichtsaugenden Schmetterlingen nachgewiesen werden. Erstere (Deilephila euphorbiae, Macroglossum stellatarum und croatica, Vanessa, C-Album, V. antiopa, V. urticae, Pieris napi, P. rapae, Papilio podalirius, P. machaon) vermögen Rohrzucker zu spalten mit Hilfe einer im Speichel und Mitteldarmsaft enthaltenen Invertase. — Diastase, Lipase, Protease fehlen im Darmsaft. Als Ort der Resorption wurde der Mitteldarm festgestellt. Bei den Formen ohne Nahrungsaufnahme (Dicranura vinula, Lymantria monacha) kommen alle Raupenfermente (also auch die Invertase) in Fortfall. Der Darm fungiert lediglich als Reservoir für den Puppenkot.     

Electrocardiographic study of rat fetuses exposed to ethylene glycol monomethyl ether (EGME)

The widely used industrial solvent ethylene glycol monomethyl ether (EGME) is teratogenic to rats and mice, inducing a variety of heart and major vessel abnormalities. In the present study, electrocardiography was used to evaluate heart function in day 20 rat (Sprague-Dawley) fetuses from mothers treated on gestation days 7-13 (sperm = day 1) with 0, 25, or 50 mg/kg EGME by gavage in 10 ml/kg water. The increased incidence of fetuses with cardiovascular malformations (primarily right ductus arteriosus and ventricular septal defect) and abnormal electrocardiograms (EKG) was dose dependent. The most prevalent EKG abnormality was a prolonged QRS wave. Mean QRS intervals were not significantly increased by EGME exposure, but there were significantly more litters in the 50-mg/kg EGME group that had one or more fetuses with QRS complexes of 40 msec or longer. The enhanced duration and the appearance of the aberrant QRS's suggested the presence of an intraventricular conduction delay in these fetuses. Heart rate and other EKG characteristics such as the P wave or P-R and Q-T intervals were not significantly affected by exposure to EGME. There did not appear to be an association between abnormal EKG's and fetal heart dysmorphology.