The structural basis of riboflavin binding to Schizosaccharomyces pombe 6,7-dimethyl-8-ribityllumazine synthase

Riboflavin is an essential cofactor in all organisms. Its direct biosynthetic precursor, 6,7-dimethyl-8-ribityllumazine, is synthesised by the enzyme 6,7-dimethyl-8-ribityllumazine synthase. Recently, we have found that the enzyme from Schizosaccharomyces pombe binds riboflavin, the final product of the pathway with a relatively high affinity with a KD of 1.2 microM. Here, we report on the crystal structure of lumazine synthase from S. pombe with bound riboflavin and compare the binding mode with those of the substrate analogue inhibitor 5-nitro-6-(D-ribitylamino)-2,4(1H,3H)-pyrimidinedione and of the product analogue 6-carboxyethyl-7-oxo-8-ribityllumazine. In all complexes the pyrimidinedione moieties of each respective ligand bind in a very similar orientation. Binding of riboflavin additionally involves a stacking interaction of the dimethylbenzene moiety with the side-chain of His94, a highly conserved residue in all lumazine synthases. The enzyme from Bacillus subtilis showed a KD of at least 1 mM whereas the very homologous enzyme from Saccharomyces cerevisiae had a comparable KD of 3.9 microM. Structural comparison of the S. cerevisiae, the S. pombe, and the mutant enzymes suggests that fine tuning of affinity is achieved by influencing this stacking interaction.     

Insulin-regulated trafficking of dual-labeled glucose transporter 4 in primary rat adipose cells

In isolated rat adipose cells, physiologically relevant insulin target cells, glucose transporter 4 (GLUT4) subcellular trafficking can be assessed by transfection of exofacially HA-tagged GLUT4. To simultaneously visualize the transfected GLUT4, we fused GFP with HA-GLUT4. With the resulting chimeras, GFP-HA-GLUT4 and HA-GLUT4-GFP, we were able to visualize for the first time the cell-surface localization, total expression, and intracellular distribution of GLUT4 in a single cell. Confocal microscopy reveals that the intracellular proportions of both GFP-HA-GLUT4 and HA-GLUT4-GFP are properly targeted to the insulin-responsive aminopeptidase-positive vesicles. Dynamic studies demonstrate close similarities in the trafficking kinetics between the two constructs and with native GLUT4. However, while the basal subcellular distribution of HA-GLUT4-GFP and the response to insulin are indistinguishable from those of HA-GLUT4 and endogenous GLUT4, most of the GFP-HA-GLUT4 is targeted to the plasma membrane with little further insulin response. Thus, HA-GLUT4-GFP will be useful to study GLUT4 trafficking in vivo while GFP on the N-terminus interferes with intracellular retention.     

Effect of long-term treatment with recombinant bovine somatotropin and estradiol on hormone concentrations and ovulatory response of superovulated cattle

The objective was to assess effects of long-term treatment with recombinant bovine somatotropin (bST) and estradiol-17beta (E2) on the number of follicles that ovulated in response to FSH. Non-lactating Holstein and Jersey cows (Trial 1, n=27) and Angus cows and heifers (Trial 2, n=35) received two ear implants of E2 and biweekly injections of bST in a 2 x 2 arrangement of treatments. Estradiol implants were removed 74.6 +/- 1.1 d after insertion and 18.1 +/- 0.9 d after the last biweekly injection of bST. Cows were stimulated with FSH-P beginning 2 d after removal of E2 implants, and PGF2alpha (PGF) was given on the third day of FSH treatment. Ovaries were collected to determine the number of CL at 1 to 2 wk after treatment with PGF. In Trial 2 only, cattle were inseminated at estrus and embryos were collected 6 to 8 d later. Implants of E2 increased (P < 0.01) serum E2 8-fold initially and E2 was still elevated 5-fold at removal of implants. Injections of bST increased (P < 0.01) serum growth hormone (GH) 15-fold and insulin-like growth factor-I (IGF-I) 3-fold. In Trial 1, number of CL was increased by the combination of bST+E2 (P < 0.01). In Trial 2, E2 increased the number of CL (P < 0.05), and bST increased the number of total ova and transferable embryos (P < 0.01). We conclude that long-term treatment with bST and E2 may interact to enhance follicular development and ovulatory response to FSH.     

Efficient plant regeneration of Mesembryanthemum crystallinum via somatic embryogenesis

 An efficient plant regeneration procedure has been established from hypocotyl explants of the common ice plant, Mesembryanthemum crystallinum L, a halophytic leaf succulent that exhibits a stress-induced switch from C3 photosynthesis to crassulacean acid metabolism (CAM). Somatic embryos were initiated and developed up to globular and heart stages in Murashige and Skoog (MS) media supplemented with 3% sucrose, 0.6% bacto-agar, 80 mM NaCl, 5 μM 2,4-D and 1 μM kinetin. High frequency regeneration occurred when somatic embryos were germinated on media that lacked 2,4-D. High cytokinin treatment suppressed normal growth of embryos and favored abnormal embryo proliferation. Without growth regulators, regenerated plants rooted on MS medium with 100% efficiency. Mature, regenerated plants were fertile and morphologically identical to seed-derived plants. Received: 29 April 1999 / Revision received: 2 July 1999 · Accepted: 12 July 1999     

The Role of the δ GABA(A) Receptor in Ovarian Cycle-Linked Changes in Hippocampus-Dependent Learning and Memory

The δ subunit of the GABA_AR is highly expressed in the dentate gyrus of the hippocampus where it mediates a tonic extrasynaptic inhibitory current that is sensitive to neurosteroids. In female mice, the expression level of the δ subunit within the dentate gyrus is elevated in the diestrous relative to estrous phase of the estrous cycle. Previous work in our lab found that female δ-GABA_AR KO mice showed enhanced hippocampus-dependent trace but normal hippocampus-independent delay fear conditioning. Wild-type females in this study showed a wide range of freezing levels, whereas δ-GABA_AR KO mice expressed only high levels of fear. We hypothesized that the variability in the wild-type mice may have been due to estrous cycle-mediated changes in the expression of the δ-GABA_AR, with low levels of freezing in mice that were in the diestrous phase when dentate gyrus tonic inhibition is high. In the present study we tested this hypothesis by utilizing contextual, delay, and trace fear conditioning protocols in mice that were trained and tested in either the diestrous or estrous phases. Consistent with our hypothesis, we found a significant impairment of hippocampus-dependent learning and memory during diestrus relative to estrus in wild-type mice and this impairment was absent in δ-GABA_AR mice. These findings argue that the δ-GABA_AR plays an important role in estrous cycle-mediated fluctuations in hippocampus-dependent learning and memory.     

Drugs inhibiting the renin - angiotensin system

There are several approaches for interfering with the renin-angiotensin system. Antibodies against renin angiotensins I and II (AI and AII) have not been consistently successful in the past, probably because of nonspecific effects; however, recent purification of renin now makes this approach more promising. Renin inhibitors include pepstatin and analogs, lipids and phospholipids, and renin-substrate analogs. Pepstatin and analogs are the most potent and specific but they are not orally active. The phospholipids are the most effective in vivo but their specificity is yet to be established. No renin-substrate analogs have been developed that have biologically significant effects. Some of the most potent and specific agents available for interfering with the renin-angiotensin system are the AII-receptor antagonists. While these compounds effectively prevent the actions of AII, they suffer from several severe deficiencies: partial agonist activity, short duration of action, and lack of oral activity. The recent development of angiotensin-converting enzyme ACE) inhibitors that are orally active has provided the greatest degree of clinical success for inhibitors of the renin-angiotensin system and, consequently, the impetus to develop still better compounds. Captopril (SQ 14,225) is the prototype ACE inhibitor, being highly potent and specific with no other demonstrated pharmacological activity. Captopril is effective in all forms of human and animal models of hypertension except mineralocorticoid hypertension, which requires concomitant diuretic therapy. Because ACE is the same enzyme as kininase II, the enzyme that degrades kinins, the possibility exists that kinins are involved in the cardiovascular action of captopril, although this prospect is unlikely.     

Cell surface labeling of glucose transporter isoform GLUT4 by bis-mannose photolabel. Correlation with stimulation of glucose transport in rat adipose cells by insulin and phorbol ester

A new impermeant photoaffinity label has been used for identifying cell surface glucose transporters in isolated rat adipose cells. This compound is 2-N-4(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannos-4- yloxy)-2- propylamine. We have used this reagent in combination with immunoprecipitation by specific antibodies against the GLUT4 and GLUT1 glucose transporter isoforms to estimate the relative abundance of these two transporters on the surface of the intact adipose cell following stimulation by insulin and phorbol 12-myristate 13-acetate (PMA). In the basal state, GLUT4 and GLUT1 are both present at the cell surface but GLUT4 is more abundant than GLUT1. In response to insulin, GLUT4 increases 15-20-fold and GLUT1 increases approximately 5-fold while 3-O-methyl-D-glucose transport is stimulated 20-30-fold. By contrast, PMA only induces a approximately 4-fold increase in GLUT4 while GLUT1 increases approximately 5-fold to the same level as seen with insulin. In addition, PMA stimulates 3-O-methyl-D-glucose transport approximately 3-fold to only 13% of the insulin-stimulated state. Thus GLUT4 is the major glucose transporter isoform under all conditions, and it is selectively and markedly enriched in response to insulin but not PMA which increases GLUT1 and GLUT4 equally. Furthermore, stimulation of glucose transport activity correlates closely with the appearance of GLUT4 on the cell surface in response to both insulin and PMA but does not correlate with the sum of GLUT1 and GLUT4 appearance. These results suggest that GLUT4 may be inherently more active than GLUT1 due to a higher TK (turnover/Km).     

Determinations of adipose cell size and number in suspensions of isolated rat and human adipose cells

The osmic acid fixation-Coulter electronic counter method described for determining adipose cell size and number in intact adipose tissue fragments has been modified for use with suspensions of isolated rat and human adipose cells. Mean cell sizes in tissue fragments and isolated cell suspensions prepared from the same tissue are virtually identical in rats of various weights. No statistically significant difference in mean adipose cell size between tissue and isolated cell suspension was observed in human adipose tissue although the variability was much greater than in rat tissue. The distribution of cell sizes among replicate samples is more uniform in the isolated cell preparations, possibly reflecting the considerably larger quantities of tissue used in preparing isolated cells than in determining cell size and number directly from tissue fragments. An example of the utility of the modified method during routine metabolic studies with isolated rat epididymal adipose cells is described; isolated cells of increasing size can be obtained from rats of increasing body weight, or from the separated distal and proximal portions of the fat pads of rats of the same weight.