Comparison of uptake kinetics in freshly isolated suspensions and short-term primary cultures of rat hepatocytes

The apparent kinetics of uptake of various model substrates were examined for hepatocytes in suspension and primary culture up to 72 h. The ability of hepatocytes to take up taurocholate and ouabain was decreased in culture. Vmax for uptake of both substrates diminished rapidly with increasing time in culture. An increase in Km was observed in cultures 6 h after plating, but there was no further change with prolongation of culture time. The decrease of uptake of taurocholate and ouabain during culture may be due to the reduction in the number of transport carriers plus a decrease of affinity of the carrier to substrates. The nonsaturable component of cadmium uptake was much reduced in cultured cells compared with the suspensions. The saturable process was lower in 6 h culture but increased to a level comparable with the fresh cells at longer culture time. No significant change was found in the Km between suspensions and cultures. Uptake of alpha-aminoisobutyric acid was greater in culture while that of 3-O-methyl-D-glucose was relatively stable but about one-half that found in cell suspension. Thus, uptake of two substrates, taurocholate and ouabain, is clearly compromised with increasing time in primary culture, while uptake of the other substrates does not reflect such a dramatic decrease. It is therefore apparent that the cell preparation of choice in uptake studies depends on the substrate and the nature of the experiments.     

Synthesis of N-carboxyalkyl and N-aminoalkyl functionalized dipeptide building units for the assembly of backbone cyclic peptides.

To improve the assembly of backbone cyclic peptides, N-functionalized dipeptide building units were synthesized. The corresponding N-aminoalkyl or N-carboxyalkyl amino acids were formed by alkylation or reductive alkylation of amino acid benzyl or tert-butyl esters. In the case of N-aminoalkyl amino acid derivatives the aldehydes for reductive alkylation were obtained from N,O-dimethyl hydroxamates of N-protected amino acids by reduction with LiAlH4. N-carboxymethyl amino acids were synthesized by alkylation using bromoacetic acid ester and the N-carboxyethyl amino acids via reductive alkylation using aldehydes derived from formyl Meldrums acid. Removal of the carboxy protecting group leads to free N-alkyl amino acids of very low solubility in organic solvents, allowing efficient purification by extraction of the crude product. These N-alkyl amino acids were converted to their tetramethylsilane-esters by silylation with N,O-bis-(trimethylsilyl)acetamide and could thus be used for the coupling with Fmoc-protected amino acid chlorides or fluorides. To avoid racemization the tert-butyl esters of N-alkyl amino acids were coupled with the Fmoc-amino acid halides in the presence of the weak base collidine. Both the N-aminoalkyl and N-carboxyalkyl functionalized dipeptide building units could be obtained in good yield and purity. For peptide assembly on the solid support, the allyl type protection of the branching moiety turned out to be most suitable. The Fmoc-protected N-functionalized dipeptide units can be used like any amino acid derivative under the standard conditions for Fmoc-solid phase synthesis.     

A familial mutation in the testis-determining gene SRY shared by both sexes

A familial mutation in SRY, the gene coding for the testis-determining factor TDF, was identified in an XY female with gonadal dysgenesis, her father, her two brothers and her uncle. The mutation consists of a T to C transition in the region of the SRY gene coding for a protein motif known as the high mobility group (HMG) box, a protein domain known to confer DNA-binding specificity on the SRY protein. This point mutation results in the substitution, at amino acid position 109, of a serine residue for phenylalanine, a conserved aromatic residue in almost all HMG box motifs known. This F109S mutation was not found in 176 male controls. When recombinant wildtype SRY and SRY^F109S mutant protein were tested in vitro for binding to the target site AAC AAAG, no differences in DNA-binding activity were observed. These results imply that the F109S mutation either is a rare neutral sequence variant, or produces an SRY protein with slightly altered in vivo activity, the resulting sex phenotype depending on the genetic back-ground or environmental factors.This paper is dedicated by G. S. to Professor Ulrich Wolf on the occasion of his 60th birthday     

Peripheral chemoreflex responsiveness is increased at elevated levels of carbon dioxide after episodic hypoxia in awake humans.

We hypothesized that the acute ventilatory response to hypoxia is enhanced after exposure to episodic hypoxia in awake humans. Eleven subjects completed a series of rebreathing trials before and after exposure to eight 4-min episodes of hypoxia. During the rebreathing trials, subjects initially hyperventilated to reduce the partial pressure of carbon dioxide (Pet(CO(2))) below 25 Torr. Subjects then breathed from a bag containing normocapnic (42 Torr), low (50 Torr), or high oxygen (140 Torr) gas mixtures. During the trials, Pet(CO(2)) increased while a constant oxygen level was maintained. The point at which ventilation began to rise in a linear fashion as Pet(CO(2)) increased was considered to be the ventilatory recruitment threshold. The ventilatory response below and above the recruitment threshold was determined. Ventilation did not persist above baseline values immediately after exposure to episodic hypoxia; however, Pet(CO(2)) levels were reduced compared with baseline. In contrast, compared with baseline, the ventilatory response to progressive increases in carbon dioxide during rebreathing trials in the presence of low but not high oxygen levels was increased after exposure to episodic hypoxia. This increase occurred when carbon dioxide levels were above but not below the ventilatory recruitment threshold. We conclude that long-term facilitation of ventilation (i.e., increases in ventilation that persist when normoxia is restored after episodic hypoxia) is not expressed in awake humans in the presence of hypocapnia. Nevertheless, despite this lack of expression, the acute ventilatory response to hypoxia in the presence of hypercapnia is increased after exposure to episodic hypoxia.     

Targeting of porin to the outer membrane of Escherichia coli. Rate of trimer assembly and identification of a dimer intermediate

Porin, a transmembrane protein in the outer membrane of Escherichia coli, exists in a trimeric structure which is not dissociated during sodium dodecyl sulfate-polyacrylamide gel electrophoresis at 25 degrees C. This unusual stability was utilized in the study of the conformational changes which accompany the targeting of porin to the outer membrane. A delay of 16-44 s between completion of synthesis of a monomer and its assembly into a trimer was found from the ratio of monomers to trimers found in exponentially growing cells. Pulse-chase experiments showed that rapid processing of precursor OmpF molecules was followed by assembly into sodium dodecyl sulfate-resistant oligomers with a half-time of 20 s at 30 degrees C. An intermediate in assembly was isolated by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis below 10 degrees C and was identified as a metastable dimer.