Investigations on vitamin D esters synthesized in rats. Turnover and sites of synthesis

1. The tissue contents of vitamin D alcohol and ester were estimated in rats 5, 10, 24, 48 and 72hr. after peroral administration of [1-(3)H]cholecalciferol. 2. The total vitamin D in liver decreased in an exponential fashion from 19% of the dose at 5hr. to 0.6% at 72hr., but the ester content remained at a relatively constant low value from 5hr., so that by 72hr. it represented 67% of the total vitamin D. Vitamin D ester in kidney increased slowly to 48hr., but by 72hr. it was only 10% of the total vitamin D. 3. The small intestine, unlike liver and kidney, contained a higher content of vitamin D ester 10hr. after administration than at later times, and it is postulated that some vitamin D was esterified during absorption from the alimentary tract. 4. Plasma contained vitamin D ester at all time intervals, and it is suggested that ester found in liver and kidney could have been transported to these sites in the blood. 5. Thoracic-duct lymph was found to transport 43% of a peroral dose of vitamin D in 12hr., of which 1.4% was esterified. The fatty acid components of the lymph vitamin D ester, determined by two-dimensional thinlayer chromatography, were mainly palmitate (31%), stearate (25%), oleate (16%) and linoleate (16%). This pattern was similar to that previously found in liver.     

Transposon mutagenesis of baculoviruses: analysis of TFP3 lepidopteran transposon insertions at the FP locus of nuclear polyhedrosis viruses

We report the complete sequences of two representatives of the TFP3 transposable element family of the lepidopteran, Trichoplusia ni. These elements were isolated as insertions mobilized from the Lepidopteran host genome into two closely related nuclear polyhedrosis viruses (NPV) during infection. Both elements inserted within the 500-bp FP locus of the respective viral genomes (map units 36.0 to 37.0), causing a distinctive plaque morphology phenotype and the loss of a 25-kDa viral-specific protein. Both insertions occurred at the identical TTAA target site in the respective genomes, in the same relative orientation, and are flanked by 15-bp imperfect inverted repeats. The inserted elements interrupt the 25K open reading frame (ORF). One of these FP mutants undergoes spontaneous reversion. Sequence analysis at the excision site of a spontaneous revertant demonstrates that the TFP3 elements are capable of precise excision, restoring the expression of the 25-kDa protein. We also compare the sequences of the 25K genes of the Autographa californica and Galleria mellonella viruses (AcMNPV and GmMNPV, respectively). The 25K gene sequences diverge in five areas, resulting in an additional EcoRV and TaqI site within the GmMNPV 25K gene, and extension of the ORF for an additional 2 amino acids at the C-terminus of the predicted GmMNPV 25 kDa protein. The phenomenon of transposon mutagenesis of Baculovirus genomes provides a unique opportunity for analysis of transposon mobility.     

Subunit structure of the erythropoietin receptor

Chemical cross-linking of the red blood cell hormone, erythropoietin (Epo), to its receptor on erythroid cells has revealed the presence of two proteins closely associated with Epo, but the relationship between these two proteins is controversial. Using the cross-linking reagents disuccinimidyl suberate and dithiobissuccinimidyl propionate, we show that 125I-Epo can be specifically conjugated in a complex of 224kDa using mouse fetal liver cells, bone marrow cells, and Friend virus-induced splenic erythroblasts as demonstrated by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels under nonreducing conditions. Under reducing conditions, the 224-kDa complex appeared as two Epo conjugates of 136 kDa and 119 kDa, and these bands were also observed to a variable extent in some nonreducing gels. Disulfide linking of the 136-kDa and 119-kDa bands was confirmed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis run under nonreducing followed by reducing conditions. With increasing time of 125I-Epo binding to Friend virus erythroblasts in the presence of sodium azide to inhibit receptor internalization, the 136-kDa and 119-kDa bands seen under reducing conditions increased markedly in intensity, whereas the 224-kDa band seen under nonreducing conditions declined. These results suggest that the 224-kDa Epo conjugate is inefficiently solubilized under nonreducing conditions following prolonged periods of Epo binding. A single class of saturable, high affinity receptors for Epo on each of the cell types tested is demonstrated. It is concluded that the two disulfide-linked Epo-binding proteins which can be independently cross-linked to Epo form a single ligand binding site.     

Application of a fiber-matrix model to transport in renal tubules

The effects of tight junction structure on water and solute fluxes across proximal tubular epithelium were examined with fiber-matrix equations previously derived by Curry and Michel (1980. Microvascular Research. 20:96-99). Using plausible estimates of tight junction fiber length and width the model predicts solute (Ps) and water permeability (Lp) coefficients that agree with the measured values. When fiber-matrix and pore models were compared for physiologically relevant ranges of matrix void fraction (80-98%) and pore radii (0-20 A), the fiber-matrix model predicted a 10-fold higher Lp/Ps ratio. Lp/Ps was most sensitive to small changes in tight junction structure when void fractions exceeded 90%. Void fractions of 96.5% and 97.1% predicted previously measured values for Lp and solute permeabilities in rat and rabbit proximal tubules. These values are consistent with void fractions and permeabilities of artificial membranes. The fiber-matrix tight junction model was incorporated into a model of reabsorption from the rat proximal tubule developed by Weinstein (1984). American Journal of Physiology. 247:F848-F862.) A void fraction of 98% predicted the experimental results for isosmotic reabsorption driven by active transport. Changing void fraction over the range of 97-99% produced a 50-75% change in predicted volume reabsorption with active transport. According to the fiber-matrix model: (a) solute permeabilities alone cannot be used to predict Lp, (b) previously measured solute permeabilities in the proximal tubule are compatible with significant water reabsorption through a water-permeable tight junction, and (c) hydraulic and solute permeabilities may be sensitive to small changes in tight junction fiber length and diameter or ionic strength within the tight junction.