Smoking and cancer: smoking cessation.

Smoking is the single most important cause of cancer. The risk of developing cancer is reduced by stopping smoking and decreases substantially after five years. Reduction in smoking must be central to any programme aimed seriously at the prevention of cancer. An individual approach, based in primary care, has the potential to bring about modest but important reductions in risk. Many randomised trials have shown the effectiveness of various smoking cessation interventions in primary care. Given resource limitations in primary care, individual effort should be focused on those at highest risk who are motivated to stop smoking. A population strategy has considerable advantages over the high risk approach as the potential for reducing morbidity and mortality in the whole population is much greater. The government must acknowledge its major responsibility; the outstanding example of its failure to do this is its persistent refusal to ban outright all forms of advertising and promotion of tobacco. There is clear evidence that a ban would contribute to a reduction in smoking prevalence and especially in the uptake of smoking by children.     

A comparative study of EPR spin trapping and cytochrome c reduction techniques for the measurement of superoxide anions

Superoxide anions (O₂^.−) generated by the reaction of xanthine with xanthine oxidase were measured by the reduction of cytochrome c and by electron paramagnetic resonance (EPR) spectroscopy using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Studies were performed to determine the relative sensitivities of these two techniques for the measurement of O₂^.−. Mixtures of xanthine, xanthine oxidase, DMPO generated two adducts, a transient DMPO-OOH and a smaller but longer-lived DMPO-OH. Both adducts were inhibited by superoxide dismutase (SOD), demonstrating they originated from O₂^.−, and were also significantly decreased when the experiments were performed using unchelated buffers, suggesting that metal ion impurities in unchelated buffers alter the formation or degradation of DMPO-adducts. O₂^.−, generated by concentrations of xanthine as low as 0.05 μM, were detectable using EPR spin trapping. In contrast, mixtures of xanthine, xanthine oxidase, and cytochrome c measured spectrophotometrically at 550 nm demonstrated that concentrations of xanthine above 1 μM were required to produce measurable levels of reduced cytochrome c. These studies demonstrate that spin trapping using DMPO was at least 20-fold more sensitive than the reduction of cytochrome c for the measurement of superoxide anions. However, at levels of superoxide generation where cytochrome c provides a linear measurement of production, EPR spin trapping may underestimate radical production, probably due to degradation of DMPO radical adducts.     

Studies on the production of human parathyroid hormone by recombinant Escherichia coli

Expression of human parathyroid hormone, hPTH(-1-84), by Escherichia coli N4830: pEX-PPTH was studied in controlled bioreactors. The hPTH is expressed as a fusion protein under control of the bacteriophage p_R promoter. In batch runs, low biomass concentrations but high specific hPTH productivities were obtained with complex TY (bactotryptone and yeast extract) medium whereas high biomass concentration and low specific productivities were found when fructose was used instead of bactotryptone (YF medium). The preinduction temperature was always 30°C; the temperature shift to induce production of fusion protein was varied from 36 to 42°C. Formation of hPTH passed a pronounced maximum as a function of induction temperature when using YF medium. However, the optimum temperature shift was 38°C for both media used. For this temperature increase both media yielded about the same volumetric hPTH productivity (approx. 30 mg hPTH/l per hour). By applying a fedbatch strategy for the YF medium, the productivity of the recombinant protein could be further increased more than fourfold. Compared to shake-flask experiments, the hPTH yield could be increased by a factor larger than 20.     

Two Voltage-Gated,Calcium Release Channels Coreside in the Vacuolar Membrane of Broad Bean Guard Cells

Voltage-gated, Ca2+ release channels have been characterized at the vacuolar membrane of broad bean guard cells using patch clamps of excised, inside-out membrane patches. The most prevalent Ca2+ release channel had a conductance of 27 pS over voltages negative of the reversal potential (Erev) (cytosol referenced to vacuole), with 5,10, or 20 mM Ca2+ as the charge carrier on the vacuolar side and 50 mM K+ on the cytosolic side. The single-channel current saturated at ~2.6 pA. The relative permeability of the channel was in the range of a Pca2+:Pk+ ratio of 6:1. Divalent cations could act as charge carriers on the vacuolar side with a conductance series of Ba2+ > Mg2+ > Sr2+ > Ca2+ and a selectivity sequence of Ca2+ [approximately equals to] Ba2+ [approximately equals to] Sr2+ > Mg2+. The channel was gated open by cytosol-negative (physiological) transmembrane voltages, increases in vacuolar Ca2+ concentration, and increases in the vacuolar pH. The channel was potently inhibited by the Ca2+ channel blockers Gd3+ (half-maximal inhibition at 10.3 [mu]M) and nifedipine (half-maximal inhibition at 77 [mu]M). The stilbene derivative 4,4[prime]-diisothiocyano-2,2[prime]-stilbene disulfonate was also inhibitory (half-maximal inhibition for a 4-min incubation period at 6.3[mu]M). The 27-pS channel coresides in individual guard cell vacuoles with a less frequently observed 14-pS Ca2+ release channel that had similar, although not identical, voltage dependence and gating characteristics and a lower selectivity for Ca2+ over K+. The requirement for two channels with a similar function at the vacuolar membrane of guard cells is discussed.     

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.     

Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.