Isolation and identification of seven metabolites of 25-hydroxydihydrotachysterol3 formed in the isolated perfused rat kidney: a model for the study of side-chain metabolism of vitamin D

The in vivo metabolism of dihydrotachysterol3, an analogue of vitamin D3 and a potent calcemic factor, has been studied in the rat. This in vivo metabolism is compared to the in vitro metabolism of 25-hydroxydihydrotachysterol3 in the perfused rat kidney. Using mass spectrometry and ultraviolet spectroscopy, we have identified seven novel metabolites derived from 25-hydroxydihydrotachysterol3. The seven compounds represent intermediates on two renal pathways (24-oxidation and 26,23-lactone formation) also observed for 25-hydroxyvitamin D3. No evidence was found for the renal synthesis of a 1-hydroxylated metabolite of 25-hydroxydihydrotachysterol3 analogous to the hormone 1,25-dihydroxyvitamin D3. Two of the compounds formed in vitro, 24,25-dihydroxydihydrotachysterol3 and 25-hydroxydihydrotachysterol 26,23-lactone, were also formed in vivo. In vivo studies also revealed the formation of two other unidentified metabolites which are presumed to be formed nonrenally and may be calcemic factors. This work shows that dihydrotachysterol3 metabolism is complex and probably utilizes the same side-chain enzymes as vitamin D3. In addition, our work also confirms that intermediates postulated to lie on pathways to 26,23-lactone in the vitamin D3 series are also formed for the side chain in dihydrotachysterol3.     

Developmental modulation of a glial cell-associated glycoprotein, 5B12, in an insect, Acheta domesticus

The expression of an insect (Acheta domesticus) adult glial cell-specific antigen, 5B12 undergoes major changes during development. The 5B12 antigen is detected as early as 20-25% of embryonic development, when immunoreactivity is distributed throughout the periphery, present at the luminal surface of epithelial cells which compose developing limb buds, sensory appendages, and the body cavity. The antigen is also localized on the cell surface of neural elements within commissural tracts in the embryonic CNS. 5B12 is secreted extracellularly in the periphery, where it is associated with the embryonic basal lamina in developing cercal sensory appendages. Luminal surface expression is transient, and disappears by 95% of embryonic development. As development proceeds, 5B12 distribution becomes more restricted, so that in the adult the antigen is predominantly associated with specific glial elements within the nervous system where it occurs as a specialized component of the extracellular matrix. The 5B12 antigen is also associated with discrete central and peripheral fiber tracts. Antigen 5B12 is present in whole embryos and in the adult CNS as a Mr 185-kDa glycoprotein. Distinct carbohydrate moieties with chondroitin sulfate-like properties are situated on the 5B12 epitope. Thus the glia-associated 5B12 macromolecule has the characteristics of a small proteoglycan. Based upon features of its distribution, pattern of spatiotemporal expression, and biochemical properties, it is speculated that 5B12 participates in events related sequentially to the development and the function of the insect nervous system.     

Distinct synthetic and structural characteristics of proteoglycans produced by cultured artery smooth muscle cells of atherosclerosis-susceptible pigeons

Proteoglycan (PG) metabolism by aortic smooth muscle cell cultures derived from atherosclerosis-susceptible White Carneau (WC) and -resistant Show Racer (SR) pigeons was compared using [35S]sodium sulfate and [3H]serine or [3H]glucosamine as labeling precursors. Chondroitin sulfate (CS) PG and dermatan sulfate (DS) PG were the major PG secreted into the medium by both cell types. Total PG production, whether measured by incorporation of radiolabel into either core protein or glycosaminoglycan chains, was consistently lower in WC compared to SR cultures at several time points. This difference was due in part to lower (30-37%) PG synthesis in WC cells, but degradation of newly synthesized PG was an important contributor. A pulse-chase study indicated that of the total radiolabeled PG present at time O, only 47% was present at 24 h in WC cultures compared to 88% in SR cultures. The large CS-PG appeared to be the primary target for degradation in WC cells, and this selective processing resulted in a higher DS-PG:CS-PG ratio in these cultures. Structural studies indicated similar core protein and glycosaminoglycan chain sizes within a PG type for both cell types. PG monomer composition differed, however, by a higher sulfation of WC CS-PG compared to SR CS-PG and by a disaccharide sulfation position favoring 6-sulfation in WC PG and 4-sulfation in SR PG.     

The use of limited proteolysis to probe interdomain and active site regions of beta-galactosidase (Escherichia coli)

Limited proteolysis by pancreatic elastase (EC 3.4.21.36) and chymotrypsin (EC 3.4.21.1) was used to study the domain structure and active site of beta-galactosidase (EC 3.2.1.23) (Escherichia coli). Treatment with elastase resulted in a rapid cleavage between residues Ala-732 and Ala-733. No inactivation accompanied this cleavage suggesting that this bond is in a hinge region of the protein. Some slow cleavages beyond the initial one were observed to occur and were accompanied by inactivation. Treatment of beta-galactosidase with chymotrypsin resulted in cleavages first between Trp-585 and Ser-586 and then between Phe-601 and Cys-602. The first of these cleavages resulted in total inactivation of beta-galactosidase. The presence of monovalent ions or isopropyl-beta-D-thiogalactopyranoside protected against the cleavages but when Mg2+ or Mn2+ was present in the reaction mixture, the bond between Trp-585 and Ser-586 was more susceptible to the action of chymotrypsin. These data demonstrate that the conformation of beta-galactosidase around Trp-585 and Ser-586 is dramatically affected by the binding of ions and isopropyl-beta-D-thiogalactopyranoside. The mutant M15 beta-galactosidase, which is missing residues 11 through 41 and is an inactive dimer rather than an active tetramer, was found to be much more labile to proteases than native beta-galactosidase, but the same initial cleavages were found to occur. In addition, trypsin cleaved the M15 protein between Arg-431 and Trp-432 while native beta-galactosidase was stable to trypsin.     

cDNA sequence of human beta-preprotachykinin, the common precursor to substance P and neurokinin A

The nucleotide sequence of cDNA encoding the human substance P precursor, beta-preprotachykinin (beta-PPT), has been determined. The source of mRNA was a human laryngeal carcinoid tumour that contained a high concentration of immunoreactive substance P. The human beta-PPT polypeptide is 129 amino acids long and contains regions encoding substance P and neurokinin A, each flanked by basic amino acid residues. Residues 72-107 of the human beta-PPT polypeptide encode the sequence of neuropeptide K, an N-terminally extended form of neurokinin A recently isolated from porcine brain.     

Esterase genes in parallel composite cross barley populations

The California population of Composite Cross V of barley was used as the source of three subpopulations that were started from generations 10, 20 and 30, respectively, and were grown in parallel environmental conditions in Cambridge for eight generations. Outcrossing rates (0.2%) were even lower than in the California material, and heterozygotes were correspondingly rare, so that the populations were essentially mixtures of homozygous lines. Four esterase loci that were polymorphic in the base Composite Cross V remained so in all the derived populations, but showed considerable changes in allelic frequency over time, particularly at two of the genes. Multilocus analysis showed that strong directional changes occurred in all three populations, but they were not consistent. One particular genotype became predominant in the population derived from generation 10, whereas in the other two populations it was a genotype with different alleles at the Est1 and Est3 loci that rose to frequencies of more than 50%. Strong directional selection undoubtedly occurred in these populations, but did not cause parallel changes in esterase gene frequencies. These data do not facilitate a discrimination between the alternative explanations of hitchhiking or multilocus selection at these loci.     

Chromosomal damage induced in human lymphocytes by low doses of D-T neutrons

Unstable chromosome aberrations induced by in vitro irradiation with zero plus seven low doses of 14.8 MeV D-T neutrons in the range 3.55-244 mGy have been analysed in human peripheral blood lymphocytes. In order to obtain the required large numbers of scored cells for such low doses, fourteen laboratories participated in the experiment. The dose responses for dicentrics, excess acentrics and total aberrations, fitted well to the Y = alpha D model. The alpha coefficient of yield for dicentrics, 1.60 +/- 0.07 X 10(-2) Gy-1, compares well with the values obtained in previous studies with D-T neutrons at somewhat higher doses. Results from a previous collaborative study using 250 kVp X-rays over a comparable dose range indicated the possible existence of a threshold below 50 mGy. In the present study there is no clear evidence for neutrons for such a threshold. However, the data were insufficient to permit the rejection of a possible threshold below approximately 10 mGy.     

Light Activation of Pyruvate,Pi Dikinase and NADP-Malate Dehydrogenase in Mesophyll Protoplasts of Maize : Effect of DCMU, Antimycin A, CCCP, and Phlorizin

Pyruvate,Pi dikinase (PPDK, EC 2.7.9.1) and NADP-malate dehydrogenase (MDH, EC 1.1.1.82) were activated in the light and inactivated following a dark treatment in mesophyll protoplasts of maize. DCMU (up to 33 micromolar), an inhibitor of noncyclic electron transport, inhibited activation of MDH much more strongly than it did PPDK. Antimycin A (6.6-33 micromolar), an inhibitor of cyclic photophosphorylation, inhibited the activation of PPDK (up to 61%), but had little or no effect on activation of MDH. Carbonyl cyanide m-chlorophenylhydrazone (0.2-2 micromolar) and nigericin (0.4 micromolar), uncouplers of photophosphorylation, inhibited activation of PPDK while stimulating the activation of MDH. Phlorizin (0.33-1.7 millimolar), an inhibitor of the coupling factor for ATP synthesis, strongly inhibited activation of PPDK but only slightly effected light activation of MDH. These results suggest that noncyclic electron flow is required for activation of NADP-MDH and that photophosphorylation is required for activation of PPDK.     

Microwave-field-driven acoustic modes in DNA.

The direct coupling of a microwave field to selected DNA molecules is demonstrated using standard dielectrometry. The absorption is resonant with a typical lifetime of 300 ps. Such a long lifetime is unexpected for DNA in aqueous solution at room temperature. Resonant absorption at fundamental and harmonic frequencies for both supercoiled circular and linear DNA agrees with an acoustic mode model. Our associated acoustic velocities for linear DNA are very close to the acoustic velocity of the longitudinal acoustic mode independently observed on DNA fibers using Brillouin spectroscopy. The difference in acoustic velocities for supercoiled circular and linear DNA is discussed in terms of solvent shielding of the nonbonded potentials in DNA.     

Stimulator requirements for primed alloreactive T cells: macrophages and dendritic cells activate T cells across all genetic disparities

The cellular requirements for stimulating primed alloreactive T cells have been investigated. In vitro-primed secondary alloreactive cells, long-term lines, and Ly 1+2- noncytolytic clones which reacted with allo-H-2K, D, or Mls (M locus) antigens were tested. The data indicated that a specialized antigen-presenting cell such as a macrophage or a dendritic cell was required for stimulating primed alloreactive cells across all the genetic disparities tested. B and T lymphocytes were ineffective stimulators. The stimulator requirement for secondary and Ly 1+2- clone responses was heterogeneous, since both macrophages and dendritic cells were effective stimulators. Thus, the allostimulator requirement for inducing proliferation and mediator secretion by the primed T-cell populations closely paralleled the requirement for stimulating unprimed populations. The only exception found was the peritoneal washout population, which did not stimulate a primary response but did stimulate secondary responses. The failure of peritoneal macrophages to stimulate a primary response was shown to be due to an inhibitory pathway which did not occur when the responding population was alloantigen primed.