Factors responsible for the formation of different N-alkylated porphyrins in rat liver microsomal systems exposed to norethindrone. The role of 3 alpha-hydroxysteroid dehydrogenase.

Incubation of rat liver microsomes with norethindrone and a NADPH-generating system leads to the formation of one N-alkylated porphyrin (green pigment, GP1). Administration of this steroid to male rats in vivo results in the formation of three more-polar green pigments (GP2, 3 and 4). A cytosolic protein (green-pigment converting protein) has been purified from rat liver that, when added to liver microsomal mixtures containing norethindrone (0.03 mM) and a NADPH-generating system, results in the formation of all four green pigments (GP1, 2, 3 and 4). Field-desorption mass spectrometry of the purified green pigments gave protonated molecules, [M + H]+, at m/z 905 for GP1, m/z 909 for GP2, m/z 925 for GP3 and 4. The Mr of the purified cytosolic protein on SDS/polyacrylamide-gel electrophoresis or gel filtration was 37000. Polyacrylamide-gel isoelectric focusing gave a pI value of 5.9. Antibodies raised in rabbits against this protein, after preincubation with rat liver cytosol, subsequently prevented the formation of the more-polar norethindrone-induced green pigments (GP2, 3 and 4). The purified protein in the presence of either NADH or NADPH catalysed the reduction of delta 4-ring-reduced norethindrone, 5 alpha-oestran-17 alpha-ethynyl-17 beta-ol-3-one and, with the appropriate cofactor, the oxidation and reduction of steroids lacking the ethynyl function, e.g. androsterone or dihydrotestosterone. Indomethacin inhibited the reduction of dihydrotestosterone by this protein with an I50 (concn. causing 50% inhibition) value of 4.9 microM. From its physical and enzymic properties it is concluded that green-pigment converting protein is the same as 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50).     

Differential processing of Asn-linked oligosaccharides on pituitary glycoprotein hormones: implications for biologic function

Luteinizing hormone, follicle-stimulating hormone, and thyroid-stimulating hormone from pituitary and chorionic gonadotropin from placenta are a family of glycoproteins, each consisting of an and subunit. Within an animal species, the subunit of all four hormones contains the identical amino acid sequence, while each subunit is distinct and confers biologic specificity to the hormone dimer. Despite sharing common subunits, these hormones bear Asn-linked oligosaccharides which differ in structure. Whereas chorionic gonadotropin contains exclusively neutral and sialylated oligosaccharides, the pituitary hormones bear neutral, sialylated, sulfated, and sialylated/sulfated structures. The sulfated oligosaccharides are unique in structure and are more prevalent on certain pituitary hormones, indicating that the synthesis of these unusual oligosaccharides is tightly regulated. The differences in oligosaccharide structures in conjunction with the highly specific endocrine responses elicited by these hormones, suggest an important functional role for the oligosaccharides, such as metabolic clearance, control of hormone response, modulation of hormone potency, and/or intracellular sorting of hormones into separate secretory granules.     

Numerical solution of a nonlinear advance-delay-differential equation from nerve conduction theory

A functional differential equation which is nonlinear and involves forward and backward deviating arguments is solved numerically. The equation models conduction in a myelinated nerve axon in which the myelin completely insulates the membrane, so that the potential change jumps from node to node. The equation is of first order with boundary values given at t=±. The problem is approximated via a difference scheme which solves the problem on a finite interval by utilizing an asymptotic representation at the endpoints, cubic interpolation and iterative techniques to approximate the delays, and a continuation method to start the procedure. The procedure is tested on a class of problems which are solvable analytically to access the scheme's accuracy and stability, then applied to the problem that models propagation in a myelinated axon. The solution's dependence on various model parameters of physical interest is studied. This is the first numerical study of myelinated nerve conduction in which the advance and delay terms are treated explicitly.Supported in part by NSF Grant MCS8301724 and by a Biomedical Research Support Grant 2SO7RR0706618 from NIH     

Mechanisms of cytoskeletal regulation: functional and antigenic diversity in human erythrocyte and brain beta spectrin

A study of human erythrocyte and brain spectrin with particular emphasis on the beta subunits revealed a structural homology but functional dissimilarity between these two molecules. Six monoclonal antibodies raised to human erythrocyte beta spectrin identify three of the four proteolytically defined domains of erythrocyte beta spectrin. Five of these monoclonal antibodies cross-react with human brain spectrin. None of a previously identified set of alpha erythrocyte spectrin monoclonal antibodies [Yurchenco et al: J Biol Chem 257:9102, 1982] reacted with brain spectrin. A domain map generated by limited tryptic digestion shows that brain spectrin is composed of proteolytically resistant domains analogous to erythrocyte spectrin, but the brain protein is more basic. The binding of brain spectrin to erythrocyte ankyrin, both in solution and on erythrocyte IOVs, yielded an association constant approximately 100 time weaker than for erythrocyte spectrin. The binding of azido-calmodulin under native conditions was specific for the erythrocyte beta subunit but was not calcium dependent. In contrast, azido-calmodulin bound only to the alpha subunit of brain spectrin in a calcium-dependent manner. The similarity of structure but modified functional characteristics of the brain and erythrocyte beta spectrins suggest that these proteins serve different cellular roles.     

Relationship between intermediate filaments and microfilaments in cultured fibroblasts: evidence for common foci during cell spreading

Spreading and fully spread chick embryo fibroblasts (CEF) were examined by double-label fluorescence microscopy using the actin-specific probe rhodamine-phalloidin and an antibody directed against CEF intermediate filaments (IF). During midspreading, a striking relationship became discernible: statistical analysis showed that approximately half of the cell population exhibited one or more phase-dense, phalloidin-binding nodules that appeared to act as foci from which IF diverged. Coincidence between actin-containing structures and IF was not limited to these centers; IF could also frequently be seen running in close parallel arrays with stress fibers. Ultrastructural analysis confirmed the presence of non-membrane-bound out-pocketings along the length of stress fibers from which 10-nm IF diverged. These structures varied in size and shape, and displayed a dense, fine fibrillar appearance. IF and microfilaments (MF) were distinguished by size and by decoration of MF with myosin subfragment-1. Other IF-MF interactions were seen in cells of all stages: IF were observed to loop through stress fibers, most frequently at the cell margins. In colchicine-treated cells, IF became redistributed into cables that often ran parallel and appeared to merge with stress fibers. Cytochalasin D-treated CEF exhibited loose aggregates of actin-containing material that appeared to be associated with IF. These results suggest the possibility of an interaction between actin-containing structures and IF, particularly during cell spreading in cultured fibroblasts.     

Growth Regulation In Multilayered Cultures of Human Diploid Fibroblasts: the Roles of Contact, Movement and Matrix Production

Abstract. Early subcultures of human embryonic lung fibroblasts are exceptional, as they grow far beyond confluence before growth ceases: the stationary dish may well contain 3-10 monolayer equivalents. Maximal growth rates, however, occur at about one-sixth confluence when doubling times are 15-20 hr; a density at which cell contacts begin to become frequent. the fact that a slowing down of growth is first apparent at such low densities argues against this regulation being due to diffusion effects. Confirmation of the role of short-range or contact interactions in growth regulation comes from an experiment using mixed cultures of fibroblasts: this shows that growth inhibition is not carried by medium-borne influences but depends on short-range (<1 mm) interactions. Evidence that cells can escape the effects of such contact interactions and so divide comes from time-lapse studies of dense cultures: there is a burst of motility soon after a fresh-medium change, which is followed by a burst of mitosis × 20 hr later. A medium change to conditioned medium supplemented with 10% foetal calf serum leads to neither the burst of motility nor the subsequent burst of mitosis, although this medium is better able to support the growth of sparse cells than is fresh medium. Data are also presented to show that the amount of collagen deposited in superconfluent cultures affects their growth: the stimulation of collagen production with ascorbic acid leads to an unexpectedly low stationary cell density and rather less movement in the culture. This result suggests that the collagen stabilizes cell contacts that are responsible for growth inhibition. the question of why these cells grow more slowly as density increases cannot be answered directly by these experiments; nevertheless, the results suggest that cell contact affects the permeability of the cell membrane to medium.     

Role and location of NAD malic enzyme in thermogenic tissues of Araceae

This work was done to discover how those nonphotosynthetic tissues of the Araceae that become thermogenic release, as CO2, carbon recently fixed by phosphoenolpyruvate carboxylase. Extracts of clubs of the spadix of Arum maculatum showed no activity for phosphoenolpyruvate carboxykinase and low activities of NADP malic enzyme. NAD malic enzyme activity in the above extracts and in those of thermogenic tissues of other Araceae was appreciable. Analysis of homogenates of clubs of Typhonium giraldii by differential centrifugation and sucrose gradients showed that NAD malic enzyme was confined to mitochondria. Centrifugation of mitochondria after freezing and thawing left all the NAD malic enzyme in the supernatant. NAD malic enzyme in isolated, intact mitochondria was completely latent, and was completely protected from exogenous trypsin. The responses of this latency and protection to different concentrations of Triton X-100 suggested that none of the NAD malic enzyme was accessible from either the outside or the intermembrane space of the mitochondria. Treatment of excised clubs of A. maculatum with 2-N-butylmalonate largely prevented the development of the rapid respiration responsible for thermogenesis, and severely inhibited dark fixation of 14CO2. The conclusion is that in mature clubs of the Araceae phosphoenolpyruvate is converted to malate in the cytosol by phosphoenolpyruvate carboxylase and NAD malate dehydrogenase, and that this malate then enters the mitochondrial matrix where it is converted to pyruvate by NAD malic enzyme.     

Fast atom bombardment: evidence of disproportionation and recombination of a synthetic porphyrin in the matrix

In addition to dimerization and polymerization of samples as previously suggested, it appears that during FAB-MS, reactions in the sample matrix can occur to yield new compounds that are recombinations of molecular fragments. This type of reaction may be especially critical to the integrity of peptide sequencing using FAB, since the reactions cited in this report involve the formation and rupture of amides or peptide bonds.     

Effects of oral propranolol and exercise protocol on indices of aerobic function in normal man

The exercise responses to two different progressive, upright cycle ergometer tests were studied in nine healthy, young subjects either with no drug (ND) or following 48 h or oral propranolol (P) (40 mg q.i.d.). The ergometer tests increased work rate by 30 W either every 30 s or every 4 min. Propranolol caused a significant (p less than 0.05) reduction in peak oxygen uptake (VO2) during both the 30-s and 4-min tests (30-s ND, 3949 +/- 718 mL X min-1 (means +/- SD); 30-s P, 3408 +/- 778 mL X min-1; 4-min ND, 4058 +/- 409 mL X min-1; 4-min P, 3725 +/- 573 mL X min-1). There was no difference between 30-s ND and 4-min ND for peak VO2. The ventilatory anaerobic threshold was not significantly different between any test (30-s ND, 2337 +/- 434 mL O2 X min-1; 30-s P, 2174 +/- 406 mL O2 X min-1; ND, 2433 +/- 685 mL O2 X min-1; 4-min P, 2296 +/- 604 mL O2 X min-1). The VO2 at which blood lactate had increased by 0.5 mM above resting levels was significantly lower than the ventilatory anaerobic threshold for the 4-min ND (1917 +/- 489) and the 4-min P (1978 +/- 412) tests, but was not different for the 30-s ND and 30-s P tests. At exhaustion in the progressive tests, the blood PCO2 was higher (p less than 0.05) in both 30-s tests than 4-min tests.(ABSTRACT TRUNCATED AT 250 WORDS)     

Partial NH2- and COOH-terminal sequence and cyanogen bromide peptide analysis of Escherichia coli sn-glycerol-3-phosphate acyltransferase

The sn-glycerol-3-phosphate acyltransferase from Escherichia coli, an integral membrane protein whose activity is dependent on phospholipids, was purified to near homogeneity (Green, P. R., Merrill, A. H., Jr., and Bell, R. M., (1981) J. Biol. Chem. 256, 11151-11159). Determination of a partial NH2-terminal sequence and the COOH terminus permitted alignment of the polypeptide on the sequenced sn-glycerol-3-phosphate acyltransferase structural gene (Lightner, V. A., Bell, R. M., and Modrich, P. (1983) J. Biol. Chem. 258, 10856-10861). Processing of the sn-glycerol-3-phosphate acyltransferase is apparently limited to the removal of the NH2-terminal formylmethionine. Thirteen of 27 possible cyanogen bromide peptides predicted from the DNA sequence were purified, characterized, and assigned to their location in the primary structure. Three peptides located at positions throughout the sequence were partially sequenced by automated Edman degradation. The partial sequence analysis of the homogeneous sn-glycerol-3-phosphate acyltransferase is fully in accord with the primary structure inferred from the DNA sequence.