Refinement of the solution structure of the DNA hexamer 5'd(GCATGC)2: combined use of nuclear magnetic resonance and restrained molecular dynamics

The solution structure of the self-complementary DNA hexamer 5'd(GCATGC)2 comprising the specific target site for the restriction endonuclease Sph 1 is investigated by using nuclear magnetic resonance spectroscopy and restrained molecular dynamics. All the nonexchangeable proton resonances are assigned sequentially, and from time-dependent nuclear Overhauser enhancement measurements a set of 158 approximate interproton distances are determined. These distances are used as the basis of a structure refinement using restrained molecular dynamics in which the interproton distances are incorporated into the total energy function of the system in the form of an effective potential term. Two restrained molecular dynamics simulations are carried out, starting from classical B- and A-DNA [atomic root mean square (rms) difference 3.3 A]. In both cases convergence is achieved to essentially identical structures satisfying the experimental restraints and having a root mean square difference of only 0.3 A between them, which is within the rms fluctuations of the atoms about their average positions. These results suggest that the restrained molecular dynamics structures represent reasonable approximations of the solution structure. The converged structures are of the B type and exhibit clear sequence-dependent variations of helical parameters, some of which follow Calladine's rules and can be attributed to the relief of interstrand purine-purine clash at adjacent base pairs. In addition, the converged restrained dynamics structures appear bent with a radius of curvature of approximately 20 A. This bending appears to be due almost entirely to the large positive base roll angles, particularly at the Pyr-Pur steps. Further, the global and local helix axes are not coincident, and the global helix axis represents a superhelical axis which the bent DNA, when extended into an "infinite" helix by repeated translation and rotation, wraps around.     

Cadmium-induced insulin release does not involve changes in intracellular handling of calcium

A possible interaction between Cd2+ and Ca2+ as a component in Cd2+-induced insulin release was investigated in beta cells isolated from obese hyperglycemic mice. The glucose stimulated Cd2+ uptake was dependent on the concentration of sugar. This uptake was sigmoidal with a Km for glucose of about 5 mM and was suppressed by both 50 microM of the voltage-activated Ca2+ channel blocker D-600 and 12 mM Mg2+. In the presence of 8 mM glucose 5 microM Cd2+ evoked a prompt and sustained stimulatory response, corresponding to about 3-fold of the insulin release obtained in the absence of the ion. Whereas 5 microM Cd2+ was without effect on the glucose-stimulated 45Ca efflux in the presence of extracellular Ca2+, 40 microM inhibited it. At a concentration of 5 microM, Cd2+ had no effect on the resting membrane potential or the depolarization evoked by either glucose or K+. In the absence of extracellular Ca2+ there was only a modest stimulation of 45Ca efflux by 5 microM Cd2+. Studies of the ambient free Ca2+ concentration maintained by permeabilized cells also indicate that 5 microM Cd2+ do not mobilize intracellularly bound Ca2+ to any great extent. On the contrary, at this concentration, Cd2+ even suppressed inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release. The present study suggests that Cd2+ stimulates insulin release by a direct mechanism which does not involve an increase in cytoplasmic free Ca2+ concentration.     

Characterization of the inositol 1,4,5-trisphosphate-induced Ca2+ release in pancreatic beta-cells.

Pancreatic beta-cells isolated from obese-hyperglycaemic mice released intracellular Ca2+ in response to carbamoylcholine, an effect dependent on the presence of glucose. The effective Ca2+ concentration reached was sufficient to evoke a transient release of insulin. When the cells were deficient in Ca2+, the Ca2+ pool sensitive to carbamoylcholine stimulation was equivalent to that released by ionomycin. Unlike intact cells, cells permeabilized by high-voltage discharges failed to generate either inositol 1,4,5-triphosphate (InsP3) or to release Ca2+ after exposure to carbamoylcholine. However, the permeabilized cells released insulin sigmoidally in response to increasing concentrations of Ca2+. Also in the absence of functional mitochondria these cells exhibited a large ATP-dependent buffering of Ca2+, enabling the maintenance of an ambient Ca2+ concentration corresponding to about 150 nM even after several additional pulses of Ca2+. InsP3, maximally effective at 6 microM, promoted a rapid and pronounced release of Ca2+. The InsP3-sensitive Ca2+ pool was rapidly filled and lost its Ca2+ late after ATP depletion. The transient nature of the Ca2+ signal was not overcome by repetitive additions of InsP3. It was possible to restore the response to InsP3 after a delay of approx. 20 min, an effect which had less latency after the addition of Ca2+. These latter findings argue against degradation and/or desensitization as factors responsible for the transiency in InsP3 response. It is suggested that Ca2+ released by InsP3 is taken up by a part of the endoplasmic reticulum (ER) not sensitive to InsP3. On metabolism of InsP3, Ca2+ recycles to the InsP3-sensitive pool, implying that this pool indeed has a very high affinity for the ion. The presence of functional mitochondria did not interfere with the recycling process. The ER in pancreatic beta-cells is of major importance in buffering Ca2+, but InsP3 only modulates Ca2+ transport for a restricted period of time following immediately upon its formation. Thereafter the non-sensitive part of the ER takes over the continuous regulation of Ca2+ cycling.     

N-Deacetylation of 2-acetamido-2-deoxy-hexosecontaining oligosaccharides and polysaccharides by calcium in liquid ammonia

N-Deacetylation of 2-acetamido-2-deoxy-hexose residues is accomplished in liquid ammonia containing calcium. Oligosaccharides, lacto-N-fucopentaose II and lacto-N-difucohexaose I, containing 3,4-disubstitutedN-acetylhexosamine residues are quantitativelyN-deacetylated. When applied to polysaccharides, however, only partialN-deacetylation was achieved.Author for correspondence. AXRD     

Immunocytochemical localization of aminopeptidase N on the cell surface of isolated porcine thyroid follicles

Summary The ultrastructural location of aminopeptidase N on the cell surface of isolated porcine thyroid follicle cells was studied with immunocytochemistry using antibodies against intestinal aminopeptidase N and protein A-colloidal gold. Gold particles, indicating immunoreactivity, were selectively attached to the apical cell surface. Occasionally, there was a sparse labelling of the basal cell surface. In follicles kept at 4° C most gold particles at the apical cell surface appeared as clusters, with each gold particle situated at a constant distance of about 20 nm from the membrane surface. The gold particles were concentrated on the membranes of microvilli, in comparison to the smooth (intermicrovillar) portions of the apical plasma membrane. In follicles incubated at 37° C for 5–180 min gold particles were slowly internalized by predominantly smooth-surfaced micropinocytic vesicles and subsequently appeared in colloid droplets and lysosomes. Gold particles were not observed in Golgi cisternae. TSH did not appear to influence the rate of internalization. TSH-induced pseudopods were unlabelled.Our electron-microscopic observations confirm previous immunofluorescence-microscopic evidence that aminopeptidase N is selectively expressed in the apical plasma membrane domain in the thyroid follicle cell. Furthermore, aminopeptidase N appears to be distributed in microdomains within the apical plasma membrane. Earlier indications of molecular differences between the pseudopod membrane and the apical plasma membrane proper are further emphasized.This study was supported by Grant No 12X-537 from the Swedish Medical Research Council     

Aminoglycoside suppression at UAG, UAA and UGA codons in Escherichia coli and human tissue culture cells

Summary We have compared the suppression of nonsense mutations by aminoglycoside antibiotics inEscherichia coli and in human 293 cells. Six nonsense alleles of the chloramphenicol acetyl transferase (cat) gene, in the vector pRSVcat, were suppressed by growth in G418 and paromomycin. Readthrough at UAG, UAA and UGA codons was monitored with enzyme assays for chloramphenicol acetyl transferase (CAT), in stably transformed bacteria and during transient expression from the same plasmid in human 293 tissue culture cells. We have found significant differences in the degree of suppression amongst three UAG codons and two UAA codons in different mRNA contexts. However, the pattern of these effects are not the same in the two organisms. Our data suggest that context effects of nonsense suppression may operate under different rules inE. coli and human cells.     

Occurrence of bacterivory in Cryptomonas,a common freshwater phytoplankter

Summary Bacterivory was detected by incorporation of 0.57 m diameter, fluorescent polystyrene beads and fluorescently labeled bacteria (FLB) in two cultured species of Cryptomonas (C. ovata and C. erosa), and a population of Cryptomonas sp in a humic, mesotrophic lake. Rates of ingestion and clearance were very low, and similar for the cultures and the in situ population. The in situ population incorporated 0.7–1.7 bacteria cell^-1 h^-1, thereby ingesting 0.3%–2.0% of the total bacterial numbers present in the water per day, and receiving less than 2% of its carbon content per day through bacterivory. Thus, bacterivory by Cryptomonas was quantitatively important neither as a sink for bacterial biomass, nor as a carbon source for the algal cells. Possibly, it served in the uptake of essential nutrients.     

Bacterioplankton growth, grazing mortality and quantitative relationship to primary production in a humic and a clearwater lake

Bacterial growth and grazing mortality were estimated from Mayto October in two south Swedish oligotrophic lakes, one beinga clearwater lake (water colour 5–10 mg Pt l^–1 DOC2.9–3.4 mg l^–1, Secchi disk depth 5.0–9.4m) and the other a humic, brownwater lake (water colour 105–165mg Pt l^–1, DOC 13.7–22.7mg l^–1, Secchi diskdepth 1.3–2.1 m). Specific rates of growth and grazingmortality were generally similar for both lakes. However, theabundance of bacteria was consistently 2–3 times higherin the water of the humic lake, suggesting that the total productionand consumption of bacterial cells were also higher than inthe dearwater lake. The ratio of bacterial secondary productionto primary production was higher in the humic lake than in theclearwater lake, indicating that the bacterioplankton of thehumic lake utilize allochthonous substrates, in addition tosubstrates originating from autochthonous primary production.Most of the bacterial loss in both lakes could be attributedto small protozoan grazers. This implies that allochthonousand autochthonous organic carbon fixed by bacterioplankton isless important in terms of carbon flow to higher trophic levelsthan would be expected if macrozooplankton were the dominantbacterivores, providing a more direct and efficient transferof carbon to larger organisms.     

Algesia and local responses induced by neurokinin A and substance P in human skin and temporal muscle

Neurokinin A (NKA), substance P (SP) and the two peptides combined (SP + NKA) were injected intracutaneously on the forearm and into the temporal muscle of healthy volunteers. Pain intensity, cutaneous wheal and flare responses and tenderness of the temporal muscle were quantitated. SP but not NKA induced cutaneous pain. This relates the algesic effect of SP to the specific N-terminal amino acid sequence of the peptide, not shared by NKA. NKA, however, potentiated the algesic effect of SP as SP + NKA induced a significantly prolonged cutaneous pain sensation. Both peptides induced wheals, but only SP induced flare. These results confirm previous studies relating wheal formation to the identical C-terminal amino acid sequence of the two peptides and flare reaction to the N-terminal part of SP. Injections into the temporal muscle did not cause pain or tenderness.