The entrapment of [14C]ascorbic acid in human erythrocytes

Radioactively labelled ascorbic acid and dehydroascorbic acid, when incubated with human blood, migrate irreversibly into human red blood cells. Isolation and characterization of the moieties trapped within the cells via infrared spectroscopy established both their identities as L-ascorbic acid. Evidence in the form of the degree of in vitro entrapment of ascorbic acid as a function of the times of incubation and the effect of incubation temperature, anion recognition site inhibitor, and active transport inhibitor on the rate of entrapment support the hypothesis that ascorbic acid is oxidized on or near the surface of the red blood cell to dehydroascorbic acid which migrates through the lipid portion of the cell wall and is reduced back to ascorbic acid within the cell. The resulting L-ascorbic acid can not pass through the cell wall and is therefore entrapped.     

Mathematical analysis of two-phase mass transfer in a batch reactor for the chemical transformation of a steroid

A reactor is described for the conversion of the slightly water-soluble steroid testosterone (T) to 4-androstene-3, 17-dione (4-AD) by enzyme in the presence of excess cofactor. Since the enzyme is subject to substrate inhibition, reaction rates are strong functions of aqueous substrate concentration. High concentrations of the substrate, testosterone, per unit reactor volume are maintained within poly(dimethylsiloxane) beads that are suspended in the aqueous enzyme solution. Mass transfer (controlled by bead size, polymer to water volume ratio, enzyme loading) is used to control the degree and rate of conversion. The reactor dynamics are predicted over a wide range of reaction conditions. The product steroid is recovered in the polymeric beads from the enzyme solution.     

Ultrastructure and division behaviour of dinoflagellate chromosomes

Chromosomes of Prorocentrum triestinum and P. micans have similar substructural and morphometrical values as revealed by electron microscopy of thin sections. However, differences were found between the species in mean length, volume and numerical density of chromosomes, and the volume of the chromosome complement, the nuclear volume and the chromosome number. When examined by a whole-mount procedure both Prorocentrum species have left-handed screw-like chromosomes which end in differentiated telomeres. The chromosomes divide sequentially from one telomere towards the other, presenting a Y and finally a V configuration. At the region where each chromosome divides nascent sister chromatids are connected by two bridges. Sister chromatids have similar quantitative values when compared with each other and with the still undivided chromosome, which suggests that both replication and division take place as coupled events.Supported by CAICYT, grant 2409/83     

Coreconstitution of bacterial ATP synthase with monomeric bacteriorhodopsin into liposomes. A comparison between the efficiency of monomeric bacteriorhodopsin and purple membrane patches in coreconstitution experiments

The conditions for coreconstitution of a bacterial ATP synthase and bacteriorhodopsin into lecithin liposomes and for light driven ATP synthesis have been optimized. A rate of maximally 280 nmol ATP min-1 mg ATP synthase-1 was achieved with monomerized bacteriorhodopsin compared with a rate of up to 45 nmol ATP min-1 mg-1 found for proteoliposomes containing bacteriorhodopsin in the form of purple membrane patches. The different rates are explained by the finding that monomeric bacteriorhodopsin is more homogeneously distributed among the liposomes than the purple membrane patches. The final activities depended on both the purification method for the two proteins and the coreconstitution procedure. Furthermore, the ratio (lipid to bacteriorhodopsin to ATP synthase) could be optimized. Light-driven ATP synthesis depends also on the type of detergent used. The best result was obtained by deoxycholate. Also the relationship between proton translocation (by bacteriorhodopsin) and ATP synthesis activity was measured. A constant H+/ATP ratio was found at higher light intensities. This ratio increased strongly at lower light intensities.     

Assignment of the 13C nuclear magnetic resonance spectrum of a short DNA-duplex with 1H-detected two-dimensional heteronuclear correlation spectroscopy.

Proton-detected 1H-13C heteronuclear correlated spectroscopy [( 1H,13C]-COSY) was used to establish relations between the carbon-13 and proton nuclear magnetic resonance chemical shifts in the hexadeoxynucleoside pentaphosphate d-(GCATGC)2. Using the previously established sequence-specific proton NMR assignments, sequence-specific assignments were thus obtained for nearly all proton-bearing carbons. This approach offers a new criterion for distinguishing between the proton NMR lines of purines and pyrimidines, based on the different proton-carbon-13 coupling constants. Furthermore, the adenine ring carbon 2 has a unique carbon-13 chemical shift, which enables a straightforward identification of the adenine C2H resonances by [1H,13C]-COSY.     

Application of an immunoprecipitation procedure to the study of SV40 tumor antigen interaction with mouse genomic DNA sequences.

Simian Virus 40 (SV40) large T antigen is a DNA binding protein with high affinity for segments of the viral genome. To find out whether T antigen also binds to sequences of genomic cellular DNA we mixed T antigen and SAU 3 A restricted mouse DNA under stringent DNA binding conditions. Resulting protein-DNA complexes were immunoprecipitated using T antigen specific monoclonal or polyclonal antibodies. The DNA fragments in the immunoprecipitates were cloned in plasmid vectors. Four plasmid clones were selected for a detailed investigation of the inserted mouse DNA fragments. Nucleotide sequencing and DNase I footprint experiments showed that T antigen binds to sites in these fragments consisting of two tandemly oriented G(A)AGGC pentamers separated by AT rich spacers of different lengths. The cellular binding sites are very similar in their architecture to the SV40-DNA binding site I. The isolated cellular DNA fragments with T antigen binding sites occur only once or a few times in the mouse genome. Our data help to further define the structure of T antigen's DNA binding sites. The genetic functions of the isolated cellular DNA elements are not known.     

A complex balanced chromosomal rearrangement in repeated abortions

Summary A double balanced reciprocal translocation involving four chromosomes, t(1;19;6;14) (1p11; 19p11; 6q25; 14q21), was found in the phenotypically normal husband in a couple referred because of repeated abortions. Reciprocal translocations, t(6;14), had been transmitted by his mother, his father being apparently homozygous for a translocation comprising pairs 1 and 19-t(1;19)(1;19). The genetic consequences of this complex chromosomal rearrangement are analyzed.     

Endothelioma cells expressing the polyoma middle T oncogene induce hemangiomas by host cell recruitment

Mouse endothelioma cells expressing the polyoma middle T oncogene induced hemangiomas in a variety of species such as mice, rats, chicks, and quails. In embryos and newborn mice the hemangiomas expanded within 10-18 hr of injection, disrupting the vasculature and causing the death of the animal. In contrast, the hemangiomas formed a stable structure reminiscent of benign human hemangiomas in adult mice within 5 days. Analysis of the cells comprising the hemangioma revealed that over 95% of the endothelial cells were host derived. No induction of host cell proliferation was detected, and no endothelial mitogens were secreted by the endothelioma cells in vitro. The maintenance of the hemangioma appeared to require the continuous presence of endothelioma cells. The results indicate that these endothelioma cells act as a potent stimulating agent in the rapid formation of hemangiomas by recruiting nonproliferating host endothelial cells.