Production of recombinant hydroxylated human type III collagen fragment in Saccharomyces cerevisiae.

A recombinant hydroxylated fragment of human type III collagen has been produced in Saccharomyces cerevisiae by coordinated coexpression of a collagen gene fragment together with both the alpha- and beta-subunit genes for prolyl-4-hydroxylase (EC 1.14.11.2). The collagen fragment consisted of 255 residues of the helical domain and the complete C-telopeptide and C-propeptide domains. It was inserted under the control of the ethanol-inducible ADH2 promoter in a multicopy, TRP1-selectable, yeast expression vector, YEpFlag1. The prolyihydroxylase subunit genes were cloned on either side of a bidirectional galactose-inducible promoter in a low-copy minichromosome yeast expression vector, pYEUra3, which is URA3 selectable. Coordinated expression of the three different gene products after cotransformation into S. cerevisiae was detected by immunoblotting. Amino acid analysis of an immunoreactive collagen fraction demonstrated the presence of hydroxyproline, while the presence of a triple-helical domain in the collagen fragment was demonstrated by its resistance to pepsin proteolysis.

Comparison of continuous infusion of fentanyl to bolus dosing in neonates after surgery.

OBJECTIVE: Concern about respiratory depression may lead to underuse of postoperative narcotic analgesia in neonates. The authors compared continuous infusion of fentanyl with bolus dosing in infants after surgery to determine whether continuous infusion is associated with less respiratory depression. STUDY DESIGN: In the first phase of the study, 16 patients were randomly assigned to receive fentanyl by continuous infusion (C) or bolus dosing every 2 hours (B) in a double-blinded trial. Respiratory events were recorded. An observational pain score and saliva for cortisol concentration were obtained 2, 8, and 24 hours after beginning treatment to compare efficacy of pain control. In the second phase, 20 additional patients received fentanyl by continuous infusion in an unblinded fashion, with the same data collection, to more accurately determine the incidence of respiratory events. RESULTS: In phase 1, apnea occurred in eight of nine B patients (89%) compared with one of seven C patients (14%; P < .009), prompting termination of the randomized trial. The incidence of apnea or significant respiratory depression in the next 20 patients (phase 2) who received fentanyl by continuous infusion was 25% (5 of 20; P < .01 v B). Episodes of apnea in B patients required significantly more intervention than episodes in C patients (P < .01). However, in phase 2, more patients remained intubated and ventilated than in phase 1. Pain scores and salivary cortisol concentrations decreased over the 24-hour study period and were similar in B and C patients during both phases of the study. CONCLUSION: Continuous infusion of fentanyl at the doses studied is associated with pain control similar to that with bolus dosing at regular intervals. Although episodes of respiratory depression were less severe and less frequent for C patients, there may be an increased need for ventilator support with continuous infusion of fentanyl to achieve acceptable pain control. Providing adequate pain control to neonates in the immediate postoperative period remains a challenge.

Glutamine-glutamate exchange between placenta and fetal liver.

The hypothesis that glutamine shuttles nitrogen between placenta and fetal liver via interconversion with glutamate was explored by infusing L-[1,2-13C2]glutamine in six fetal sheep chronically catheterized for sampling of the umbilical and hepatic circulations. Fetal plasma glutamine disposal rate was 19.9 +/- 1.3 mumol.min-1.kg fetus-1. Entry of glutamine from the placenta accounted for approximately 60% of the total glutamine entry rate in fetal plasma. Glutamine was taken up by fetal liver, and 45.3 +/- 7.9% of the glutamine taken up was released as glutamate. The fetal liver released large quantities of glutamate, as evidenced by a sixfold increase in plasma glutamate concentration in the blood flowing through the left hepatic lobe and a hepatic glutamate output-to-O2 uptake molar ratio of 0.149 +/- 0.013. In conjunction with a previous study of fetal glutamate metabolism, these data demonstrate that glutamine entering the fetal circulation is converted to glutamate by the fetal liver at a rate of approximately 3-4 mumol.min-1.kg fetus-1.

Glutamate metabolism in fetus and placenta of late-gestation sheep.

Glutamate is produced by the fetal liver and taken up by the placenta. To explore the functional meaning of this exchange, the disposal rate (DR), clearance, conversion to glutamine, and decarboxylation rate of fetal plasma glutamate were studied at 129 +/- 2 days of gestation in seven fetal lambs infused via a systemic vein with L-[2,3,3,4,4-2H5]glutamate and L-[1-14C]glutamate. In two experiments, L-[1-13C]glutamate was also infused. The mean glutamate DR and clearance were 11.9 +/- 1.3 mumol.min-1.kg-1 and 200 +/- 8 ml.min-1.kg-1, respectively. The placenta extracted 88.5 +/- 0.8% of the tracer glutamate carried by the umbilical circulation and contributed to 61.3 +/- 3.2% of the glutamate DR. Most of the 14C infused as L-[1-14C]glutamate was converted to 14CO2: 37 +/- 4% by the fetus and 41 +/- 6% by the placenta. Of the labeled glutamate taken up by the placenta, 6.2 +/- 1.5% was returned to the fetus as glutamine. The glutamine-to-glutamate enrichment ratio in fetal arterial plasma was 0.066 +/- 0.008. We conclude that fetal plasma glutamate has an exceptionally high clearance because the flux of glutamate into the placenta is virtually equal to umbilical glutamate delivery rate. The main pathway of fetal plasma glutamate disposal is oxidation by placental and fetal tissues. Placental conversion of glutamate to fetal glutamine is a relatively small component of the placental metabolism of fetal glutamate.