Cell density-sensing in Dictyostelium by means of the accumulation rate, diffusion coefficient and activity threshold of a protein secreted by starved cells

The simple eukaryote Dictyostelium discoideum grows as an amoeba on leaf and soil surfaces. When starved, the amoebae aggregate and differentiate. The amoebae can also be induced to differentiate as isolated cells submerged in buffer, if the buffer contains a sufficiently high concentration of a protein (CMF) secreted by starved cells. CMF is also necessary for aggregation and differentiation on surfaces. This indicated that CMF has either an autocrine function or is part of a density-sensing system. To distinguish between these two possible functions, we first examined the rate at which CMF is accumulated and the activity threshold of cells for CMF, since both parameters will affect whether a cell can provide enough CMF to self-stimulate. We find that CMF potentiates its own accumulation, and that otherwise the accumulation rate and activity threshold are affected very little by a variety of physiological conditions. We then use diffusion calculations to show that even after many hours of continuous secretion, the CMF concentration adjacent to an isolated starved cell on a leaf or soil surface will be too low to allow differentiation, whereas an extracellular concentration of CMF sufficiently high to allow differentiation will occur when starved cells are at high densities. We find a close match between the predicted and experimentally observed density necessary for differentiation. The theoretical and observed behavior of cells at different cell densities suggests that due to its accumulation rate, diffusion coefficient, and activation threshold, CMF can function as part of a cell density-sensing system which allows Dictyostelium cells in the wild to co-ordinate their development.     

Sequence-specific changes in the metal site of ferric bleomycin induced by the binding of DNA

The binding of the iron complex of the antineoplastic glycopeptide bleomycin A2 (Fe-BLM) to calf thymus DNA and the self-complementary oligonucleotides d(CGCGCG) and d(ATATAT) has been studied using optical, EPR, and resonance Raman spectroscopies. An increase in the intensity of the bands at 365 and 384 nm is observed in the optical spectrum of Fe(III)-BLM when the drug binds to either oligonucleotide. However, in the presence of phosphate, this increase is observed only with d(CGCGCG) and not with d(ATATAT). In addition, the gmax feature in the EPR spectrum of low spin Fe(III)-BLM is narrowed in a way suggesting a reduction of possible conformers that the drug can achieve when it is bound to d(CGCGCG) or to calf thymus DNA but not when bound to d(ATATAT). When Fe(III)-BLM is bound to d(CGCGCG), changes in the resonance Raman spectrum of the metal drug complex suggest conformational changes in three of the ligands to iron: the beta-hydroxyhistidyl amide, the pyrimidine, and the axial hydroxide. In addition, the Fe-OH band undergoes narrowing, again consistent, with the reduction of conformers of the drug. No such resonance Raman changes are observed upon binding to d(ATATAT). The changes in the pyrimidine modes upon binding d(CGCGCG) to the drug are consistent with a recently proposed model (Wu, W., Vanderwall, D. E., Turner, C. J., Kozarich, J. W., and Stubbe, J. (1996) J. Am. Chem. Soc. 118, 1281-1294) of DNA recognition by activated bleomycin, HOO-Fe(III)-BLM, in which the pyrimidine moiety of the drug is important for the preferential cleavage of 5'-GpPy-3' sequences.     

Changes of relaxation times (T1, T2) and apparent diffusion coefficient after permanent middle cerebral artery occlusion in the rat: temporal evolution, regional extent, and comparison with histology

The quantitative NMR parameters T1, T2, rho, and apparent diffusion coefficient (ADC) were determined during the 7 h after middle cerebral artery occlusion in rats. In the normal caudate-putamen (CP), 869 +/- 145 ms and 72 +/- 2 ms for T1 and for T2, respectively, were found; the corresponding values for cortex were 928 +/- 117 ms and 73 +/- 2 ms. The ADC showed significant dependence on gradient direction: diffusion along x resulted in 534 +/- 53 microns 2/s (CP) and 554 +/- 62 microns 2/s (cortex), and along y in 697 +/- 58 microns 2/s (CP) and 675 +/- 53 microns 2/s (cortex). In the ischemic territory, a continuous increase over time of both relaxation times was observed in the CP, leading to an increase of 29 +/- 20% (T1) and 51 +/- 41% (T2) above control level. ADC dropped to 63 +/- 15% of control in the CP and to 74 +/- 4% of control in the temporal cortex. No significant change was noted in proton density during the observation period. Strongest ADC reduction was in the center of the ischemic territory (< or = 60% of control) surrounded by a region of lesser reduction (< or = 80% of control). During the early part of the study, the area of reduced ADC was larger than that of elevated relaxation times. Toward the end of the experiment, the area of increased relaxation times approached that of decreased ADC at < or = 80% of control. Good agreement of histological presentation of infarct with the total area of decreased ADC (< or = 80%) was demonstrated.     

Mechanism of endothelium-dependent relaxation induced by thrombin in the pig coronary artery

The present study describes the characterization of the binding properties and autoradiographic distribution of a new nonpeptide antagonist of neurotensin receptors, [3H]SR 142948A (2-[[5-(2,6-dimethoxyphenyl)-1-(4-(N-(3-dimethylaminopropyl)-N-methyl carbamoyl)-2-isopropylphenyl)-1H-pyrazole-3-carbonyl]-amino]-ad amantane-2-carboxylic acid, hydrochloride), in the rat brain. The binding of [3H]SR 142948A in brain membrane homogenates was specific, time-dependent, reversible and saturable. [3H]SR 142948A bound to an apparently homogeneous population of sites, with a Kd of 3.5 nM and a Bmax value of 508 fmol/mg of protein, which was 80% higher than that observed in saturation experiments with [3H]neurotensin. [3H]SR 142948A binding was inhibited by SR 142948A, the related nonpeptide receptor antagonist, SR 48692 (2-[[1-(7-chloroquinolin-4-yl)-5-(2,6-dimethoxyphenyl)-1H-pyrazole -3-carbonyl]amino]-adamantane-2-carboxylic acid) and neurotensin. Saturation and competition studies in the presence or absence of the histamine H1 receptor antagonist, levocabastine, revealed that [3H]SR 142948A bound with similar affinities to both the levocabastine-insensitive neurotensin NT1 receptors (20% of the total binding population) and the recently cloned levocabastine-sensitive neurotensin NT2 receptors (80% of the receptors) (Kd = 6.8 and 4.8 nM, respectively). The regional distribution of [3H]SR 142948A binding in the rat brain closely matched the distribution of [125I]neurotensin binding. In conclusion, these findings indicate that [3H]SR 142948A is a new potent antagonist radioligand which recognizes with high affinity both neurotensin NT1 and NT2 receptors and represents thus an excellent tool to study neurotensin receptors in the rat brain.     

Relation between the solidification rate of a binary alloy and the alloying-element distribution coefficient

The change in the rate of equilibrium and nonequilibrium (according to Petrov–Scheil) solidification in binary model and some real solid-solution alloys with a distribution coefficient 0.2–2.5 is comprehensively analyzed. A relation between the solidification rate, on the one hand, and growth restriction factor Q and supercooling parameter P, which are used to determine the grain sizes in as-cast alloys, is revealed.     

Spreading waves of a reduced diffusion coefficient of water in normal and ischemic rat brain

Using echo planar diffusion-weighted magnetic resonance imaging, we measured three-dimensional changes in the apparent diffusion coefficient (ADC) of water in eight contiguous coronal slices, encompassing the entire rat brain, before and after local cortical stimulation. We applied chemical (potassium chloride application; n = 6) and mechanical (needle stab; n = 4) stimulations to the right posterior parietal rat cortex. In all animals in which potassium chloride or the needle stab was applied, a region of decreased ADC values to a mean of 0.45 +/- 0.03 x 10(-5)cm2/s occurred. These reduced ADC levels appeared in the posterior parietal cortex within 1 min after cortical stimulation and the change recovered within 1 min. Then a ripple-like movement of similar changes developed across the unilateral cortex. This change was localized to the cortex and no significant ADC changes occurred in subcortical structures. The propagating speed of this movement was 3.4 +/- 0.5 mm/min. These findings are compatible with spreading depression as observed electrophysiologically. Similar ADC changes occurred in areas distinct from the ischemic lesion in 3 of 12 animals subjected to focal cerebral ischemia. This magnetic resonance method could detect spreading ADC decline if it occurred in human diseases including brain ischemia.     

A diffusion solution for the melting/dissolution of a solid at constant temperature and its use for measuring the diffusion coefficient in liquids

A general solution to the diffusion equation is obtained for the composition in two phases when one phase dissolves and the other grows at constant temperature. The relevance of the derivation to melting is discussed. The exact solution is used to examine the condition when diffusion may be considered to occur in only one phase. Experiments have been carried out using the analysis to obtain solute diffusion coefficients in liquid succinonitrile-water alloys. The method relies on an interface displacement measurement and does not require the composition to be measured as a function of position as is usually necessary with a diffusion couple.     

酸法地浸采铀多井系统中渗透系数时空演化模拟

为了解酸法地浸采铀过程中含矿含水层渗透系数时空演化规律,通过数值模拟的方法建立了由两组五点型地浸抽注井构成的二维反应动力学模型,模拟酸法地浸采铀中孔隙度-渗透系数时空演化过程,探讨含矿含水层渗透系数时空演化规律。模拟结束时模拟区域内渗透系数区间为[5.37 m/d, 14.7 m/d]。通过渗透系数时间演化结果可知,在渗流作用和化学反应前锋不断向抽液孔推进,渗透系数发生变化的区域随时间不断增大,模拟区域内渗透系数区间值随时间不断增大,模拟结束时6个注液孔周围的渗透系数演化情况差异较小;矿层内渗透系数大于初始值的范围为注液孔中心相距约3 m（占抽注半径1/10）处的圆柱体内,渗透系数最小值出现在与注液孔中心相距约5.66 m处的圆周上,含矿层内大部分区域的渗透系数均小于初始值,根据模拟结果渗透系数最小值可能出现在与注液孔距离抽注半径的1/6~1/5区域内。      

Measurement of spin-lattice relaxation times and kinetic rate constants in rat muscle using progressive partial saturation and steady-state saturation transfer

The determination of neomycin sulfate by liquid chromatography using a column packed with a poly(styrenedivinylbenzene) co-polymer and pulsed electrochemical detection on a gold electrode is described. The mobile phase consisted of an aqueous solution containing 70 g/l of sodium sulfate, 1.4 g/l of sodium 1-octanesulfonate and 50 ml/l of a 0.2 M phosphate buffer (pH 3.0). Sodium hydroxide was added post-column. The influence of the different chromatographic parameters on the separation was investigated. The method shows good linearity and repeatability and is stability indicating. A number of commercial samples was analyzed using this method and the results were compared with results obtained with the European Pharmacopoeia method and a previously described thin-layer chromatographic method.     

The apparent diffusion coefficient of water in gray and white matter of the infant brain

PURPOSE: The purpose was to obtain normal values of the apparent diffusion coefficient (ADC) in the infant brain and to compare ADC maps with T1- and T2-weighted images. METHOD: Diffusion was measured in nine infants with an ECG-gated SE sequence compensated for first-order motion. One axial slice at the basal ganglia level was investigated with the diffusion-encoding gradients in the slice-selection direction. RESULTS: On ADC maps, the corpus callosum and the optic radiations appeared dark before the onset of myelination, and the crus posterior of the internal capsule could be visualized before it appeared on T1- or T2-weighted images. In gray and white matter, the mean ADC ranged from 0.95 x 10(-9) to 1.76 x 10(-9) m2/s. In the frontal and occipital white matter, in the genu corporis callosi, and in the lentiform nucleus, the ADC decreased with increasing age. The cortex/white matter ratio of the ADC increased with age and approached 1 at the age of 30 weeks. CONCLUSION: ADC maps add information to the T1 and T2 images about the size and course of unmyelinated as well as myelinated tracts in the immature brain.     

The prokaryote-to-eukaryote transition reflected in the evolution of the V/F/A-ATPase catalytic and proteolipid subunits

The D4/S4-5 interhelical region plays a role in sodium channel fast inactivation. Examination of S4-5 primary structure in all domains suggests a possible amphipathic helical conformation in which a conserved group of small hydrophobic residues occupies one contiguous surface with a more variable complement of nonpolar and polar residues on the opposite face. We evaluated this potential structure by replacing each residue in D4/S4-5 of the rat SkM1 skeletal muscle sodium channel with substitutions having different side chain properties. Of the 63 mutations analyzed, 44 produced functional channels. P1473 was intolerant of substitutions. Nonpolar substitutions in the conserved hydrophobic region were functionally similar to wild type, while charged mutations in this region before P1473 were nonfunctional. Charged mutations at F1466, M1469, M1470, and A1474, located on the opposite surface of the predicted helix, produced functional channels with pronounced slowing of inactivation, shifted voltage dependence of steady-state inactivation, and increased rate of recovery from inactivation. The substituted-cysteine-accessibility method was used to probe accessibility at each position. Residues L1465, F1466, A1467, M1469, M1470, L1472, A1474, and F1476C were easily accessible for modification by sulfhydryl reagents; L1464, L1468, S1471, and L1475 were not accessible within the time frame of our measurements. Molecular dynamics simulations of residues A1458 to N1477 were then used to explore energetically favorable local structures. Based on mutagenesis, substituted-cysteine-accessibility method, and modeling results, we suggest a secondary structure for the D4/S4-5 region in which the peptide chain is alpha-helical proximal to P1473, bends at this residue, and may continue beyond this point as a random coil. In this configuration, the entire resultant loop is amphipathic; four residues on one surface could form part of the binding site for the inactivation particle.