Single versus repeated-course antenatal corticosteroids: outcomes in singleton and multiple-gestation pregnancies.

The objectives of this study are to compare the neonatal risks and benefits of antenatal single-course versus repeated-course corticosteroids in singleton and multiple-gestation pregnancies. A comprehensive analysis was performed of the inpatient records of all neonates admitted to our center from 1 January 1994 through 31 May 1999. The primary outcome measure was survival without chronic lung disease (CLD). Secondary outcome measures included birth weight; head circumference; interval weight ratios; respiratory disease severity; intraventricular hemorrhage rate and severity; severe retinopathy of prematurity; early infection; and hospital days. All singletons 27-32 completed weeks' gestation, and multiples 26-32 weeks' gestation, whose mothers had received betamethasone before delivery, were included. One hundred and fifteen singleton and 53 multiple-gestation infants (total 168) were stratified by multiplicity, gestational-age (< or =29 or > or =30 weeks), and number of steroid courses. Repeated courses of antenatal betamethasone were not associated with greater survival without CLD, in either singleton- or multiple-gestation infants. In singletons there was no difference in any outcome measure between groups. In multiples, the only difference was greater postnatal weight gain in the lower gestation group. Mean birth head circumference was smaller in repetitively-treated singletons < or =29 weeks. There are no clinically significant neonatal benefits of repeated-course antenatal steroids in singletons > or =27 weeks estimated gestational age (EGA) or multiple-gestation infants > or =26 weeks EGA. Prospective randomized trials of single-course versus repetitive antenatal corticosteroid therapy are warranted.

Secretion of biologically active glycoforms of bovine follicle stimulating hormone in plants.

We chose the follicle stimulating hormone (FSH), a pituitary heterodimeric glycoprotein hormone, as a model to assess the ability of the plant cell to express a recombinant protein that requires extensive N-glycosylation for subunit folding and assembly, intracellular trafficking, signal transduction and circulatory stability. A tobacco mosaic virus (TMV) based transient expression system was used to express a single-chain (sc) version of bovine FSH in the tobacco related species Nicotiana benthamiana. Preparations of periplasmic proteins from plants infected with recombinant viral RNA contained high levels of sc-bFSH, up to 3% of total soluble proteins. Consistently, in situ indirect immunofluorescence revealed that the plant cell secreted the mammalian secretory protein to the extracellular compartment (EC). By mass spectrometric analysis of immunoaffinity purified sc-bFSH derived from EC fractions, we found two species of the plant paucimannosidic glycan type, truncated forms of complex-type N-glycans. Stimulation of cAMP production in a CHO cell line expressing the porcine FSH receptor acknowledged the native-like structure of sc-bFSH and a sufficient extent of N-glycosylation required for signal transduction. Furthermore, in superovulatory treatments of mice, sc-bFSH displayed significant in vivo bioactivity, although much lower than that of pregnant mare serum gonadotropin. We conclude that plants may have a broad utility as hosts for the recombinant expression of proteins even where glycosylation is essential for function.

The N-terminal 77 amino acids from tobacco N-acetylglucosaminyltransferase I are sufficient to retain a reporter protein in the Golgi apparatus of Nicotiana benthamiana cells.

In order to investigate sequences of tobacco N-acetylglucosaminyltransferase I (GnTI), involved in targeting to and retention in the plant Golgi apparatus the cytoplasmic transmembrane stem (CTS) region of the enzyme was cloned in frame with the cDNA of the green fluorescent protein (gfp) and subsequently transiently expressed in Nicotiana benthamiana plants using a tobacco mosaic virus (TMV) based expression vector. Confocal laser scanning microscopy showed small fluorescent vesicular bodies in CTS-gfp expressing cells, while gfp alone expressed in control plants was uniformly distributed in the cytoplasm. The CTS-gfp fusion protein colocalised with immunolabelling observed by an antibody specific for the Golgi located plant Lewis a epitope. Furthermore, treatment with brefeldin A, a Golgi specific drug, resulted in the formation of large fluorescent vesiculated areas. These results strongly suggest a Golgi location for CTS-gfp and as a consequence our findings reveal that the N-terminal 77 amino acids of tobacco GnTI are sufficient to target to and to retain a reporter protein in the plant Golgi apparatus and that TMV based vectors are suitable vehicles for rapid delivery of recombinant proteins to the secretory pathway.