Solid state 13C NMR of unlabeled phosphatidylcholine bilayers: spectral assignments and measurement of carbon-phosphorus dipolar couplings and 13C chemical shift anisotropies.

The direct measurement of 13C chemical shift anisotropies (CSA) and 31P-13C dipolar splitting in random dispersions of unlabeled L alpha-phase phosphatidylcholine (PC) has traditionally been difficult because of extreme spectral boradening due to anisotropy. In this study, mixtures of dimyristoyl phosphatidylcholine (DMPC) with three different detergents known to promote the magnetic orientation of DMPC were employed to eliminate the powder-pattern nature of signals without totally averaging out spectral anisotropy. The detergents utilized were CHAPSO, Triton X-100, and dihexanoylphosphatidylcholine (DHPC). Using such mixtures, many of the individual 13C resonances from DMPC were resolved and a number of 13C-31P dipolar couplings were evident. In addition, differing line widths were observed for the components of some dipolar doublets, suggestive of dipolar/chemical shift anisotropy (CSA) relaxation interference effects. Oriented sample resonance assignments were made by varying the CHAPSO or DHPC to DMPC ratio to systematically scale overall bilayer order towards the isotropic limit. In this manner, peaks could be identified based upon extrapolation to their isotropic positions, for which assignments have previously been made (Lee, C.W.B., and R.G. Griffin. 1989. Biophys. J. 55:355-358; Forbes, J., J. Bowers, X. Shan, L. Moran, E. Oldfield, and M.A. Moscarello. 1988. J. Chem. Soc., Faraday, Trans. 1 84:3821-3849). It was observed that the plots of CSA or dipolar coupling versus overall bilayer order obtained from DHPC and CHAPSO titrations were linear. Estimates of the intrinsic dipolar couplings and chemical shift anisotropies for pure DMPC bilayers were made by extrapolating shifts and couplings from the detergent titrations to zero detergent. Both detergent titrations led to similar "intrinsic" CSAs and dipolar couplings. Results extracted from an oriented Triton-DMPC mixture also led to similar estimates for the detergent-free DMPC shifts and couplings. The results from these experiments were found to compare favorably with limited measurements made from pure L alpha PC. This detergent-based method for assigning spectra and for determining dipolar couplings and CSA in detergent-free systems should be extendable to other lipid systems. The resulting data set from this study may prove useful in future modeling of the structure and dynamics of DMPC bilayers. In addition, the fact that experiments utilizing each of the three detergents led to similar estimates for the spectral parameters of pure DMPC, and the fact that spectral parameter versus bilayer order plots were linear, indicate that the averaged conformation and dynamics of DMPC in the presence of the three detergents are very similar to those of pure L alpha DMPC.     

A three-phase pattern in growth, monoclonal antibody production, and metabolite exchange in a hybridoma bioreactor culture

The use of partial cubic spline data interpolation for the calculation of volumetric metabolite exchange rates suggested the existence of three distinct metabolic phases during bioreactor culture of a hybridoma cell line. During phase 1, a rapid amino acid uptake rate and ammonia release rate were observed. The growth rate was low and glutamine synthetase activity fell. In phase 2, maximum growth rate and minimum glutamine assimilation and ammonium production rates were observed. Attempts to corroborate the apparent ammonia assimilation in this phase using (15)NH(4)Cl resulted in low incorporation rates into alanine and glutamine. Maximum glutamine synthetase activity took place during this period. Maximum antibody production rate was observed during phase 3 during which peaks in glutamine assimilation, ammonia release, and glutamine synthetase activity were observed. The apparent existence of the three phases prompted us to carry out Northern blot analysis of glutamine synthetase RNA at appropriate times during the process. This revealed a pattern of appearance and dis-appearance of mRNA consistent with the three phases indicated by the fermentation parameters. (c) 1993 John Wiley & Sons, Inc.     

Chicken oocyte growth: receptor-mediated yolk deposition

During the rapid final stage of growth, chicken oocytes take up massive amounts of plasma components and convert them to yolk. The oocyte expresses a receptor that binds both major yolk lipoprotein precursors, vitellogenin (VTG) and very low density lipoprotein (VLDL). In the present study, in vivo transport tracing methodology, isolation of coated vesicles, ligand- and immuno-blotting, and ultrastructural immunocytochemistry were used for the analysis of receptor-mediated yolk formation. The VTG/VLDL receptor was identified in coated profiles in the oocyte periphery, in isolated coated vesicles, and within vesicular compartments both outside and inside membrane-bounded yolk storage organelles (yolk spheres). VLDL particles colocalized with the receptor, as demonstrated by ultrastructural visualization of VLDL-gold following intravenous administration, as well as by immunocytochemical analysis with antibodies to VLDL. Lipoprotein particles were shown to reach the oocyte surface by passage across the basement membrane, which possibly plays an active and selective role in yolk precursor accessibility to the oocyte surface, and through gaps between the follicular granulosa cells. Following delivery of ligands from the plasma membrane into yolk spheres, proteolytic processing of VTG and VLDL by cathepsin D appears to correlate with segregation of receptors and ligands which enter disparate sub-compartments within the yolk spheres. In small, quiescent oocytes, the VTG/VLDL receptor was localized to the central portion of the cell. At onset of the rapid growth phase, it appears that this pre-existing pool of receptors redistributes to the peripheral region, thereby initiating yolk formation. Such a redistribution mechanism would obliterate the need for de novo synthesis of receptors when the oocyte's energy expenditure is to be utilized for plasma membrane synthesis, establishment and maintenance of intracellular topography and yolk formation, and preparation for ovulation.     

ATP,pH and Mg2+ modulate a cation current in Beta vulgaris vacuoles: A possible shunt conductance for the vacuolar H+-ATPase

Macroscopic instantaneous and time-dependent currents have been measured in the vacuolar membrane of Beta vulgaris using a patch clamp configuration analogous to whole cell mode. At low cytosolic Ca²⁺ and in the absence of Mg²⁺, only an instantaneous current was observed. This current is carried predominantly by cations (P_KP_Cl 71, p_nap_cl 41 and arginine is also conducted). The instantaneous current can be activated by ATP^4– (e.g., ATP-activated mean K⁺ current density was –20 mA.m^–2 at a membrane voltage of –20 mV) and by increasing cytosolic pH and Mg²⁺ (raising Mg²⁺ from 0 to 0.4 mm induced a mean current density increase of –7 mA.m^–2 at –20 mV). Such current can be activated by simultaneous addition of putative in vivo concentrations of ATP^4–/MgATP/Mg _free ²⁺ (in the presence of bafilomycin to inhibit the vacuolar ATPase) and further modulated by cytosolic pH. With vacuolar K⁺ concentration greater than that of the cytosol, activation of the instantaneous current would mediate vacuolar K⁺ release over the range of physiological membrane voltage. It is argued that the ATP^4–-activated current, in addition to acting as a K⁺ mobilization pathway, could provide a counter-ion (shunt) conductance, allowing the two electrogenic H⁺ pumps which reside in the vacuolar membrane to acidify the vacuolar lumen.A separate time-dependent current, which was not observed at low Ca²⁺ concentrations (less than 500 nm) could also be elicited by addition of Mg²⁺ at the cytoplasmic membrane face. This current was stimulated by increasing cytoplasmic pH.The authors are grateful to the BBSRC for financial support (Grant PG87/529) and to the Royal Society (University Research Fellowship to J.M.D.). We thank C. Abbott, K. Partridge and J. Robinson for plant cultivation; A. Amtmann, A. Bertl, D. Gradmann and G. Thiel for helpful discussion.     

Crystallization of acetate kinase from Methanosarcina thermophila and prediction of its fold.

The unique biochemical properties of acetate kinase present a classic conundrum in the study of the mechanism of enzyme-catalyzed phosphoryl transfer. Large, single crystals of acetate kinase from Methanosarcina thermophila were grown from a solution of ammonium sulfate in the presence of ATP. The crystals diffract to beyond 1.7 A resolution. Analysis of X-ray data from the crystals is consistent with a space group of C2 and unit cell dimensions a = 181 A, b = 67 A, c = 83 A, beta = 103 degrees. Diffraction data have been collected from the crystals at 110 and 277 K. Data collected at 277 K extend to lower resolution, but are more reproducible. The orientation of a noncrystallographic two-fold axis of symmetry has been determined. Based on an analysis of the predicted amino acid sequences of acetate kinase from several organisms, we hypothesize that acetate kinase is a member of the sugar kinase/actin/hsp70 structural family.     

Incidence of acute symptomatic toxoplasma retinochoroiditis in south London according to country of birth.

OBJECTIVE--To determine the incidence of acute symptomatic toxoplasma retinochoroiditis presenting to ophthalmologists for patients born in Britain and elsewhere. DESIGN--Population based, cross sectional study. SETTING--11 districts in south Greater London. SUBJECTS--All patients presenting to NHS ophthalmologists with symptoms due to acute toxoplasma retinochoroiditis in 1992-3. MAIN OUTCOME MEASURE--Intraocular inflammation in association with a retinochoroidal scar, active adjoining retinitis, and IgG serum antibodies to toxoplasma. RESULTS--The estimated incidence of acute symptomatic retinochoroiditis for all people born in Britain was 0.4/100,000/year. If a mean of two symptomatic episodes per lifetime is assumed, 100 people born in Britain may be affected each year, about a fifth of the estimated 500-600 congenitally infected people born each year. CONCLUSIONS--A substantial proportion of people with acute symptomatic toxoplasma retinochoroiditis were born outside the country, and the number born in Britain was smaller than the number previously estimated to develop retinochoroidal lesions due to congenital toxoplasmosis. These findings suggest that prenatal screening for toxoplasmosis in Britain may be of limited benefit.     

The effects of hyperthermia and hyperthermia plus microwaves on rat brain energy metabolism

The effects of hyperthermia, alone and in conjunction with microwave exposure, on brain energetics were studied in anesthetized male Sprague-Dawley rats. The effect of temperature on adenosine triphosphate concentration [ATP] and creatine phosphate concentration [CP] was determined in the brains of rats that were maintained at 35.6, 37.0, 39.0, and 41.0 degrees C. At 37, 39, and 41 degrees C brain [ATP] and [CP] were down 6.0, 10.8, and 29.2%, and 19.6, 28.7, and 44%, respectively, from the 35.6 degrees C control concentrations. Exposure of the brain to 591-MHz radiation at 13.8 mW/cm2 for 0.5, 1.0, 3.0, and 5.0 min caused further decreases (below those observed for 30 degrees C hyperthermia only) of 16.0, 29.8, 22.5, and 12.3% in brain [ATP], and of 15.6, 25.1, 21.4, and 25.9% in brain [CP] after 0.5, 1.0, 3.0, and 5.0 min, respectively. Recording of brain reduced nicotinamide adenine dinucleotide (NADH) fluorescence before, during, and after microwave exposure showed an increase in NADH fluorescence during microwave exposure that returned to preexposure levels within 1 min postexposure. Continuous recording of brain temperatures during microwave exposures showed that brain temperature varied between -0.1 and +0.05 degrees C. Since the microwave exposures did not induce tissue hyperthermia, it is concluded that direct microwave interaction at the subcellular level is responsible for the observed decrease in [ATP] and [CP].