Proteoglycan metabolism associated with mouse metanephric development: morphologic and biochemical effects of beta-D-xyloside

Morphology and de novo incorporation of [35S]sulfate into proteoglycans were studied in fetal mouse kidneys at the onset of organogenesis. Branching morphogenesis and nephron development in organ culture and in vivo were associated with de novo synthesis of chondroitin-SO4 and heparan-SO4 proteoglycans. The role of proteoglycan metabolism in metanephrogenesis was then studied by analysis of the effects of p-nitrophenyl-beta-D-xylopyranoside (beta-D-xyloside) on renal development and proteoglycan metabolism. Incubation of fetal kidneys in beta-D-xyloside at concentrations of 1.0 and 0.5 mM, but not at 0.1 mM, caused inhibition of ureteric branching and markedly diminished synthesis of a large Mr 2.0 X 10(6) Da chondroitin-SO4 proteoglycan. Incorporation of [35S]sulfate was stimulated at all beta-D-xyloside concentrations, reflecting synthesis of xyloside initiated dermatan-35SO4 chains. In contrast to dramatic effects on chondroitin-SO4 synthesis and ureteric branching, beta-D-xyloside had no effect on heparan-SO4 synthesis or on development of the glomerulus and glomerular basement membrane. We thus characterize the proteoglycans synthesized early in the course of renal organogenesis and describe observations which suggest an association between metabolism of chondroitin-SO4 proteoglycan and development of the ureter.     

The acceleration of linear DNA during pulsed-field gel electrophoresis

The velocity and orientation of T4 and lambda DNA have been measured for the first 20 s during pulsed-field gel electrophoresis in order to clarify the DNA motions that occur. For a square pulse with field strength E = 10 V/cm, the velocity of lambda DNA increases gradually to 10.5 microns/s in 1.0 s, declines to 8.6 microns/s, and then rises to a plateau value of 9.3 microns/s after 4 s. T4 DNA behaves similarly, but more slowly. Parallel measurements of fluorescence-detected linear dichroism show that the DNA becomes substantially aligned with its chain axis parallel to the electrophoretic field E after the pulse is applied. The alignment also shows an overshoot, an undershoot, and a plateau comparable to those seen for velocity. When the field strength increases, both the velocity and the alignment reach their peaks more quickly. For all field strengths and both molecular weights, the velocity peak occurs when the molecular center of mass has moved 0.3 to 0.5 L, where L is the chain contour length. A qualitative model is provided.     

Variable evolutionary stability of Y chromosomal repeated sequences in the genus Mus

The study reported here is an examination of the organization and evolution of three Y chromosomal repeated sequences, designated pBC10-0.6, pBC15-1.1, and pBA33-1.8, in five closely related species of the genus Mus. The species distributions of major restriction fragment length polymorphisms produced with a panel of restriction enzymes is used to develop the phylogenetic relationships between the five species studied. However, the apparent degree of relatedness among these species varied a great deal with each of the three probes and was also highly dependent on the particular restriction enzyme used. The usefulness for phylogenetic studies of closely associated sequences varying in evolutionary stability is discussed.     

Regio- and stereoselective regulation of monooxygenase activities by isoenzyme-selective phosphorylation of cytochrome P450

The phosphorylation of the two major phenobarbital-inducible cytochrome P450 isoenzymes IIB1 and IIB2 was increased in hepatocytes by the action of the membrane permeating cAMP derivatives N6-dibutyryl-cAMP and 8-thiomethyl-cAMP. Under these conditions the dealkylation of 7-pentoxyresorufin, a selective substrate of cytochrome P450IIB1 and P450IIB2 was markedly reduced. 16 beta-Hydroxylation of testosterone which is catalyzed specifically only by cytochrome P450IIB1 and IIB2 was strongly reduced; for 16 alpha-hydroxylation which is also catalyzed by cytochrome P450IIB1 and IIB2 but additionally by 3 further cytochrome P450 isoenzymes, this reduction was less pronounced; for the oxidation of the 17 beta-hydroxyl group which besides cytochromes P450IIB1 and IIB2 is additionally catalyzed not only by other cytochromes P450 but also by 17 beta-hydroxysteroid dehydrogenase there was a clear tendency of reduction which, however, no longer reached statistical significance. Hydroxylation at other positions of testosterone which are catalyzed by other cytochrome P450 isoenzymes were not significantly changed. Hence isoenzyme-selective phosphorylation of cytochrome P450 leads to a corresponding isoenzyme-selective modulation of monooxygenase activity which holds promise to be especially important as a fast regulation of the control of genotoxic metabolites.     

Development and testing of protocols for computer-aided design of peptide drugs, using oxytocin

Considerable clinical interest in neuropeptides and peptide hormones has stimulated recent research and development of peptide-based drugs. This process differs from most classical drug discovery procedures because peptide molecules have considerable inherent flexibility. In the present paper, to identify lowest energy and metastable conformers for drug design, and to develop protocols for such studies, conformational search algorithms, incorporating empirical energy calculations, have been applied in the analysis of the peptide oxytocin. Energy minimization in torsion angle space was carried out from a variety of starting conformations, including published structures, in all-atom mode and all with distance constraints for disulphide bond formation. The energy-minimized conformations have been further optimized by a mapping method. Complementary simulations have been performed in united-atom mode and a model representing the effects of water using dummy sites has been developed and tested for this representation. Several of the preferred conformers together with de novo conformations have been used as starting points in molecular dynamics simulations; 28 low potential energy conformations were located at a temperature of 4 K. Conformations are analysed to identify hydrogen bonds, phi-psi angle distributions and the RMS values relative to the X-ray structure of deamino-oxytocin. The modelled structure of lowest energy in the molecular mechanics calculations was also that of least RMS deviation from the crystal structure; whilst structures of lower energy but larger deviation were identified by molecular dynamics techniques. A metastable structure has been identified which satisfies existing criteria for the "active form", and this model is tested by a theoretical residue-substitution technique, to provide clues on the agonist/antagonist relationship at the atomic level.     

A comparison of net and gross rates of oxygen production as a function of light intensity in some natural plankton populations and in a Synechococcus culture

In vitrorates of gross and net oxygen production were measuredas a function of light intensity in some plankton communitiescollected from Bedford Basin, Nova Scotia, and in a monoclonalculture of Synechococcus. The rate of gross oxygen productionwas measured by a technique in which the stable oxygen isotope,¹⁸O, serves as a photosynthetic tracer Net oxygen productionwas measured by automated Winkler technique. The rate of communityrespiration in the light was then determined by the differencebetween gross and net rates of oxygen production. In the naturalpopulations examined, neither gross nor net oxygen productionrates were significantly inhibited at the highest light intensitymeasured (500–800 µE m^–2 s^–1) In a samplein which the dark respiration rate was small relative to themaximal rate of production [P_max;sensu Platt et al (1980) JMar. Res., 38, 687–701] the rates of ‘light’respiration were 3 times greater. In two other communities,with high rates of dark respiration relative to P_maxthe ratesof ‘light’ respiration were closer to rates of darkrespiration. In the Synechococcus clone, both gross and netoxygen production rates were inhibited at high light intensities.Rates of ‘light’ respiration were found to varyas a function of light intensity. The greatest rates of respirationwere measured in samples incubated at light intensities thatwere just saturating (100 µE m^–2 s^–1). Therates of ¹⁴C production were also measured as a function oflight intensity The photosynthetic quotients, based on ¹⁴C productionrates and gross oxygen production rates, average 1 9     

The ATP binding site on rho protein. Affinity labeling of Lys181 by pyridoxal 5'-diphospho-5'-adenosine

We have labeled the nucleoside triphosphate-binding domain of Escherichia coli rho factor with the ATP affinity analog [3H]pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP). PLP-AMP completely inactivates the RNA-dependent ATPase activity of rho upon incorporation of 3 mol of reagent/mol of hexameric rho protein. Although the potency of PLP-AMP is enhanced when an RNA substrate such as poly(C) is present, the stoichiometry for inhibition remains the same as in the absence of poly(C). The nucleotide substrate ATP competes very effectively for the binding site and protects against PLP-AMP inactivation. A domain of rho called N2, which comprises the distal two-thirds of the molecule (residues 152-419) and encompasses the region proposed to bind ATP, is labeled specifically in the presence of poly(C). Amino acid sequence analysis of the single [3H]PLP-AMP labeled proteolytic fragment showed Lys181 to be the site of modification, suggesting that this residue normally interacts with the gamma-phosphoryl of bound ATP. These results agree with our proposed tertiary structure for the ATP-binding domain of rho that places this lysine residue in a flexible loop above a hydrophobic nucleotide-binding pocket comprised of several parallel beta-strands, similar to adenylate kinase, F1-ATPase, and related ATP-binding proteins. Parallel studies of rho structure and function by site-directed mutagenesis and chemical modification support this interpretation.