Hepatic uptake of indocyanine green and perfusion rate in rats: effect of age and albumin concentration

Liver blood flow and hepatic uptake of some indicator substances have been reported to fall with age in both rats and humans. We used an isolated liver system, which was perfused in one pass with hemoglobin free buffer, to investigate the effect of albumin concentration, buffer flow rate, and age upon hepatic clearance of the dye, indocyanine green. We measured the half-life of a bolus of indocyanine green given intravenously to male Sprague-Dawley rats aged 10 and 24 months and then examined its clearance in vitro using their isolated perfused livers. After perfusion, the livers were homogenized and separated into subcellular fractions. The mean liver weight declined significantly (young, 19.7 +/- 2.9 g vs. old, 13.9 +/- 2.6 g; p less than 0.02). In vivo the indocyanine green clearance was reduced in the aged rats (3.2 +/- 1.0 vs. 5.1 +/- 1.7 mL/min; p less than 0.05). In the isolated perfused liver system, extraction ratio showed an inverse curvilinear correlation with albumin concentration and buffer flow rate, but did not differ with age. Hepatic protein content and dye subcellular localization did not differ between the two groups. In conclusion, the fall in indocyanine green clearance in vivo is not paralleled by the ability of the organs to extract the dye in vitro, and likely reflects a decline in hepatic mass and blood flow.     

Morphogenesis in vitro of dissociated fetal rat small intestinal cells upon an open surface and subsequent to collagen gel overlay

The morphogenesis of cells dissociated from fetal rat intestine at 18 days of gestation was compared in vitro on two different substrates, tissue culture plastic and collagen gel. During the first 2 days in culture growth on both substrates was similar, resulting in the formation of layers of epithelial cells and of small lumina within them. On plastic, the cell layers contracted over time, resulting in the formation of small mounds of cells bearing on their surface small protrusions covered with epithelial cells which had densely packed microvilli on their apical surfaces. When the cultures on collagen gels were overlaid with more collagen gel, vesicles lined with epithelial cells developed. These cells were joined by junctional complexes and displayed an apical brush border which was periodic acid-Schiff and alkaline phosphatase positive. After 1 week in culture, when the vesicles reached their maximum extent, numerous epithelial cells were actively incorporating labeled thymidine and cells were being extruded into the lumen. These results demonstrate that the dissociated intestinal cells have the capacity to form intestine-like organoids in vitro and that surrounding the cells with the collagen gel allows expression of this potential. The collagen gel cultures thus undergo morphogenetic processes which can be modified by the surrounding environment and should provide a useful in vitro model system for the study of intestinal development.     

Interchromosomal Biased Gene Conversion, Mutation and Selection in a Multigene Family

A mathematical model of the effects of interchromosomal biased gene conversion, mutation and natural selection on a multigene family is developed and analyzed. The model assumes two allelic states at each of n loci. The effects of genetic drift are ignored. The model is developed under the assumption of no recombination, but the analysis shows that, at equilibrium, there is no linkage disequilibrium, which implies that the conclusions are valid for arbitrary recombination among loci. At equilibrium, the balance between mutation, gene conversion and selection depends on the ratio of the mutation rates to the quantity [s + g(2α - 1)/ n], where s is the increment or decrement in relative fitness with each additional copy of one of the alleles, g is the conversion rate, and α is a measure of the bias in favor of one of the alleles. When this quantity is large relative to the mutation rates, the allele that has the net advantage, combining the effects of selection and conversion, will be nearly fixed in the multigene family. A comparison of these results with those from a comparable model of intrachromosomal biased conversion shows that biased interchromosomal conversion leads to approximately the same equilibrium copy number as does intrachromosomal conversion of the same strength. Interchromosomal conversion is much more effective in causing the substitution of one allele by another. The relative frequencies of interchromosomal and intrachromosomal conversion is indicated by the extent of the linkage disequilibrium among the loci in a multigene family.     

Purification of d'Anjou Pear (Pyrus communis L.) Polyphenol Oxidase

Polyphenol oxidase (PPO) was extensively purified to homogeneity from d'Anjou pear (Pyrus communis L.) by extraction in the presence of the phenolic binder AG 2-X8 andTriton X-100. Chlorophyll pigment was removed by chromatography resulting in a clear, colorless enzyme extract. Purification of pear PPO was achieved after chromatography on Phenyl Sepharose CL-4B, DEAE-cellulose, and hydroxylapatite columns. Only after the columns were run at room temperature rather than at 4°C were sharp peaks and good resolution obtained. Reproducibility of the entire scheme was excellent with chromatography on the hydrophobic resin as a key to successful purification. Three separate fractions of pear PPO were homogeneous when stained for protein with the silver stain after polyacrylamide slab gel electrophoresis and corresponded to relative mobilities of 0.41, 0.43, and 0.73. The effect of dimethylsulfoxide on enzyme activity was investigated and found to increase significantly the activity of purified pear PPO over the control.     

An interaction between season of calving and nutrition on the resumption of ovarian cycles in post-partum beef cattle

Angus cows were first mated at approximately 27 months of age in 2 herds, calving 21 July to 15 September (Group E) or 9 September to 30 October (Group L). The cows were fed a high (H) or medium (M) plane of nutrition for 55 days before and 40 days after calving. There was a mean liveweight difference of 35 kg between cows in Groups EH + LH and Groups EM + LM immediately after calving and at 40 days after calving. Immediately after calving cows in Groups EH + EM were 11 kg heavier than cows in Groups LH + LM, but there was no difference at 40 days after calving. There was a significant interaction between calving time and nutrition in the return of cyclic ovarian function assessed from both interval to first oestrus and first elevated progesterone concentration. Mean intervals from calving to first oestrus were 66.7, 82.7, 56.7 and 62.3 days in Groups EH, EM, LH and LM respectively. These data demonstrate that season of calving influences resumption of ovarian cycles even at a constant high plane of nutrition and that season of calving interacts with nutrition such that effects of season are more likely to be expressed under conditions of low nutrition.     

Protonated state of methotrexate, trimethoprim, and pyrimethamine bound to dihydrofolate reductase

13C nuclear magnetic resonance (NMR) of methotrexate, trimethoprim, and pyrimethamine enriched 90% with 13C at C2 has provided a sensitive means of detecting the state of protonation of the heterocyclic rings of these inhibitors. In each case, protonation of N1 causes an upfield movement of the chemical shift of C2 by more than 6 ppm. By this method it has been shown that, at pH values up to 9.2, methotrexate is bound to bovine liver dihydrofolate reductase with N1 of the inhibitor protonated, just as in the case of the complex with reductase from Streptococcus faecium and Lactobacillus casei. Furthermore, trimethoprim bound to reductase from any of the three sources, and pyrimethamine bound to either of the bacterial reductases also have N1 protonated even at pH values up to 10. This implies that in all cases there is a strong interaction between protonated N1 of the inhibitor and the carboxylate group of the active site aspartate or glutamate. In every case pKa of the bound inhibitor is increased by several units, a finding in accord with crystallographic evidence that inhibitor bound to L. casei reductase is in a hydrophobic environment and that N1 is not hydrogen-bonded to water. It was confirmed by titration of protein fluorescence that trimethoprim has greater affinity for bacterial reductase than for vertebrate (bovine) reductase, and that this selectivity is more marked in ternary complexes in which NADPH is also bound to the active site. However, the data cited above indicate that this difference in affinities is not due to a weaker ionic interaction between protonated N1 of trimethoprim and the bovine enzyme. Instead, binding of the trimethoprim side chain to hydrophobic sites on the enzyme must provide less binding energy in the case of the mammalian enzyme.     

Interaction of AP-2, a monoclonal antibody specific for the human platelet glycoprotein IIb-IIIa complex, with intact platelets

A murine monoclonal antibody, designated AP-2, reacts specifically with the complex formed by human platelet membrane glycoproteins IIb and IIIa, but does not react at all with the individual glycoproteins. Purified AP-2 covalently coupled to Sepharose CL4B was used as an immunoadsorbent column to purify the IIb-IIIa complex from a preparation of Triton X-100-solubilized human platelet proteins. Radioiodinated AP-2 was shown to bind to a single class of sites, with 57,400 +/- 9,700 molecules bound per cell (mean +/- S.D.) at saturation and a dissociation constant (Kd) of 0.64 +/- 0.15 nM (mean +/- S.D.). Binding could not be readily reversed even after a 1-h incubation with a 100-fold excess of cold antibody. AP-2 inhibits ADP-induced binding of radiolabeled fibrinogen to gel-filtered platelets in a noncompetitive fashion, consistent with the previous observation that AP-2 also inhibits the aggregation of platelets in plasma induced by a number of physiologic agonists, including adenosine diphosphate, epinephrine, collagen, thrombin, and arachidonic acid. Using AP-2, we have obtained evidence that the IIb-IIIa complex exists in the membrane of intact nonstimulated platelets and that complex integrity is not affected by external calcium ion concentration.     

Transposable Elements in Mendelian Populations. II. Distribution of Three COPIA-like Elements in a Natural Population of DROSOPHILA MELANOGASTER

Twenty X chromosomes isolated from a natural population of Drosophila melanogaster were surveyed using in situ hybridization to determine the number and cytogenetic location of three families of transposable elements: copia, 412 and 297. We found no sites of insertions in high frequency; in fact, frequencies of specific sites for all three elements were so low that each insertion could be interpreted as being unique. This suggests that rates of transposition and deletion for these elements are very high. Our data also show a higher than expected rate of the co-occurrence of different elements at the same site on the same chromosome.