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A 235-bp DNA coding for the leech blood coagulation inhibitor, hirudin, was chemically synthesized. The synthesis involved preparation of seven long oligodeoxyribonucleotide pairs which were assembled and cloned using a rapid and simple procedure. More than half of the transformed Escherichia coli cells expressed a biosynthetic polypeptide having biological properties which were very similar to authentic hirudin from the leech Hirudo medicinalis. To achieve efficient expression, we fused the hirudin DNA to a truncated C1 repressor gene of bacteriophage lambda to create a hybrid protein. An additional methionine at the fusion point allowed the active hirudin to be cleaved off by cyanogen bromide.

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Specific transformation of a rifampicin sensitive strain of Escherichia coli to rifampicin resistance has been performed by a single, defined DNA restriction fragment carrying the genetic information for the beta subunit of E. coli RNA polymerase. In this transformation the transforming genetic character has been substituted for the corresponding recipient gene locus by recombination. The value of the described transformation system for locating genetic markers on DNA restriction fragments is discussed in comparison to previously reported in vitro systems.

Background: Meconium may cause lung injury (meconium aspiration syndromeϭMAS). Treatment is symptomatic by ventilatory support or in the worst cases extracorporal membrane oxygenation (ECMO). The pathophysiology is complex including a substantial inflammatory reaction in the lungs, but according to our results this inflammation may be systemic as well. We recently showed for the first time that meconium is a potent activator of complement (Castellheim A. et al. Pediatr. Res. 2004;55:310 -318), leading us to hypothesize that complement activation is an essential part of the pathophysiology of MAS.Methods: MAS was induced by instillation of meconium into the lungs of newborn pigs (nϭ8). To mimic the asphyxia in clinical MAS, hypoxia was induced by supplying 8% oxygen in nitrogen until base excess reached -20 mmol/l. Anaesthesia was induced by Halothane and maintained intravenously by fentanyl and midazolam. Control animals (nϭ5) received saline under otherwise identical conditions for 7 hours. Hemo-and lung dynamics were recorded. Systemic complement activation, revealed by the terminal sC5b-9 complex (TCC), and cytokines were measured in plasma samples by enzyme immunoassays. Granulocyte expression of CD18 and CD11b, as well as oxidative burst, were measured by flow cytometry.Results: Plasma TCC increased rapidly in the MAS animals (0.31Ϯ0.34 to 3.10Ϯ0.66) (AU7mL) (meanϮ2SEM), but decreased in the controls (0.55Ϯ0.88 to 0.28Ϯ0.10) (MAS vs controls: pϽ0.0005). The TCC concentration correlated closely with oxygenation-and ventilation indices (rϭ0.48 and 0.57, pϭ0.001 and Ͻ0.0005, respectively), and inversely with compliance (rϭ-0.63, pϽ0.0005); all thee reflecting severe deterioration in pulmonary function. Granulocyte oxidative burst declined significantly in the MAS animals compared with the controls (pϭ0.02) and correlated inversely with TCC (rϭ-0.37, pϭ0.02), probably reflecting a paralysis of granulocytes as part of a systemic inflammatory response. Finally, IL-6and IL-8 increased in MAS (IL-6: (26Ϯ3 to 188Ϯ80)(pg/mL) IL-8 (19Ϯ2 to 37Ϯ6)(pg/mL) animals compared to the controls (MAS vs controls: IL-6, pϭ0.002 and IL-8, pϭ0.001) Conclusion: We have for the first time demonstrated that complement is rapidly and systemically activated in experimental MAS. We suggest that this activation may induce secondary inflammatory reactions like cytokine production and oxidative burst, which contribute to the pathogenesis of MAS. Anti-complement therapy may be a rational for treatment of this disease. Objective: To investigate effects of early and late cord-clamping on short-and long-term neurologic and developmental outcome of infants born at full-term. Design: A prospective randomised study. Settings: The Perinatal Centre of the School of Medicine University of München. Subjects: 30 full-term neonates with early (Ͻ10 s) and 30 with late (3 min) cord-clamping. Intervention: Holding the infant at the level of the introitus vaginae and clamping the cord exactly 3 min after birth, or clamping the cord within 10 s of birth. Ma...