Intracellular pH electrode : Experiments on the giant squid axon

A new, easy method to produce and calibrate a 1-μm tip intracellular pH electrode is described. This antimony electrode and a micro-calomel electrode were inserted into the giant axon of Loligo pealii. The potential obtained when the axon was bathed in seawater corresponded to a pH of 7.0 ± 0.2. It was found that acidification of the external perfusate induced a drop in axoplasmatic pH leading to changes in the membrane electrical properties. Changes of resting or action potentials did not influence intracellular pH.     

Cyanoketone competition with estradiol for binding to the cytosolic estrogen receptor

Cyanoketone, an inhibitor of many steroidogenic processes, has been found to inhibit binding of estradiol to its receptor in a competitive manner. The Ki observed was 1.2 X 10(-6)M. This action may explain some of cyanoketone's effects in vivo.     

Effects of phospholipid and detergent on the substrate specificity of adrenal cytochrome P-450scc. Substrate binding and kinetics of cholesterol side chain oxidation

Cytochrome P-450scc was isolated from mitochondria of bovine adrenal cortex by hydrophobic chromatography on octyl Sepharose followed by affinity chromatography on cholesterol-7-(thiomethyl)carboxy-3 beta-acetate-Sepharose. The partially purified eluate from the octyl Sepharose resin was free of adrenodoxin and adrenodoxin reductase and displayed biphasic binding characteristics for cholesterol, cholesterol sulfate, and cholesterol acetate (CA). Chromatography of the octyl Sepharose eluate on CA-Sepharose removed extraneous proteins and resolved the cytochrome P-450scc into two fractions, each of which displayed monophasic binding with all three substrates. These fractions behaved identically with respect to their ability to bind substrates, their kinetic properties, and their rate of migration during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The dissociation constants of the cytochrome P-450scc.substrate complexes are 1.1, 2.6, and 1.3 microM for cholesterol, cholesterol sulfate, and cholesterol acetate, respectively. Addition of phospholipids isolated from adrenal cortex mitochondria or adrenodoxin had no effect on the equilibrium binding constants. Addition of Emulgen 913, however, decreased the binding affinities 10-20-fold. Emulgen 913 also inhibited the interaction of adrenodoxin with the cytochrome. An active side chain cleavage system was reconstituted with purified P-450 by addition of saturating amounts of adrenodoxin, adrenodoxin reductase, and NADPH-generating system. The apparent Km values for this reconstituted system of cholesterol, cholesterol sulfate, and cholesterol acetate are 1.8, 1.9, and 0.6 microM, respectively. Since the Km values of substrate oxidation are similar to the Kd values of the cytochrome P-450.substrate complexes, it seems likely that the binding of substrates, particularly when the side chain cleavage system is free of mitochondrial membranes, is not rate-limiting. Based on these results and electrophoretic data, it appears that one cytochrome P-450 present in adrenal mitochondria can oxidize cholesterol, its sulfate, and its acetate. This enzyme represented about 60% of the cytochrome P-450 present in the octyl Sepharose eluate. The factors responsible for the biphasic kinetics of oxidation by intact mitochondria and biphasic binding of sterol substrates by partially purified preparations of cytochrome P-450scc are still unknown.     

Structural deformation of the preterm trachea during acute distention and collapse.

The compliant airways of the premature neonate undergo episodic distention and collapse in response to changes in transmural pressure such as occur during spontaneous breathing, mechanical ventilation, and various therapeutic maneuvers. To identify and quantitate the effects of distending and collapsing transmural pressures on the structure of immature airways, tracheal segments from fetal rabbits, fixed at 0, +30, and -30 cm H2O transmural pressure, were examined using histologic and morphometric techniques. In comparison to control sections fixed at 0 cm H2O transmural pressure, application of distending pressures led to evagination of the posterior tracheal wall and significantly increased (P less than 0.05) cross-sectional area, antero-posterior diameter, circumference and muscle length, and decreased muscle thickness. Collapsed tracheal segments (-30 cm H2O) demonstrated invagination of the posterior wall and significantly (P less than 0.05) lower cross-sectional area, and antero-posterior diameter compared to the control segments; all the other parameters remained relatively unchanged. These data demonstrate extreme changes in tracheal geometry in response to the acute application of transmural pressure. From a methodological perspective, these observations suggest that fixation pressures may present significant artifact in histological analyses. Functionally, the noted deformation may lead to alterations in anatomic dead space and airway resistance, and mechanical function of the airways; all of which may compromise respiratory status in ventilated premature infant.     

The relative importance of photoperiod and temperature as cues for seasonal acclimation of thermoregulation in pouched mice (Saccostomus campestris: Cricetidae) from southern Africa

Summary The effect of short photoperiod and cold on metabolism and thermoregulation was investigated in pouched mice (Saccostomus campestris: Cricetidae) from three localities in southern Africa which experience contrasting climatic conditions. Mice were initially acclimated to long photoperiod (14L: 10D) at 25°C, followed first by a decline in photoperiod (to 10L: 14D) and then by a fall in temperature (to 10°C). Minimum observed metabolic rate (basal metabolic rate) was unaffected by the decline in photoperiod but increased significantly following cold acclimation. Because minimal thermal conductance remained constant throughout the study the increase in minimum observed metabolic rate caused a decline in lower critical temperature to around 26°C. In contrast to minimum observed metabolic rate, regulatory non-shivering thermogenesis improved significantly following the decline in both photoperiod and temperature. However, pouched mice from the warmest locality were significantly less responsive to photoperiod than those from the other two localities whose survival might depend upon their ability to accurately predict seasonal changes in temperature. Neither photoperiod nor temperature had any effect on body mass, yet pouched mice from the most arid locality, where food supply might be unpredictable, were significantly smaller and had lower total energy requirements than those from areas experiencing higher annual rainfall. These results indicate that S. campestris displays considerable geographical variation in energy requirements together with differences in the use of photoperiod as an anticipatory cue for predicting the onset of winter. These differences appear to be related to the availability of energy and the relative severity of climatic conditions in each locality.Abbreviations ANOVA analysis of variance - BMR basal metabolic rate - C _m minimal thermal conductance - M _b body mass - MOMR minimum observed metabolic rate - MWU Mann-Whitney U-test - NA noradrenaline - NST non-shivering thermogenesis - RMR resting metabolic rate - RQ respiratory quotient - T _a ambient temperature - T _b body temperature - T _1c lower critical temperature - oxygen consumption - maximum - following NA injection     

An efficient automated computer vision based technique for detection of three dimensional structural motifs in proteins.

As the number of available three dimensional coordinates of proteins increases, it is now recognized that proteins from different families and topologies are constructed from independent motifs. Detection of specific structural motifs within proteins aids in understanding their role and the mechanism of their operation. To aid in identification and use of these motifs it has become necessary to develop efficient methods for systematic scanning of structural databases. To date, methods of structural protein comparison suffer from at least one of the following limitations: (1) are not fully automated (require human intervention), (2) are limited to relatively similar structures, (3) are constrained to linear alignments of the structures, (4) are sensitive to insertions, deletions or gaps in the sequences or (5) are very time consuming. We present a method to overcome the above limitations. The method discovers and ranks every piece of structural similarity between the structures compared, thus allowing the simultaneous detection of real 3-D motifs in different domains, between domains, in active sites, surfaces etc. The method uses the Geometric Hashing Paradigm which is an efficient technique originally developed for Computer Vision. The algorithm exploits the geometrical constraints of rigid objects, it is especially geared towards recognition of partial structures in rigid objects belonging to large data bases and is straightforwardly parallelizable. Computer Vision techniques are for the first time applied to molecular structure comparison, resulting in an efficient, fully automated tool. The method has been tested in a number of cases, including comparisons of the haemoglobins, immunoglobulins, serine proteinases, calcium binding proteins, DNA binding proteins and others. In all examples our results were equivalent to the published results from previous methods and in some cases additional structural information was obtained by our method.     

Plasma lipopolysaccharide (LPS)-binding protein. A key component in macrophage recognition of gram-negative LPS.

LPS-binding protein (LBP) binds with high affinity (Kd approximately equal to 10(-9) M) to lipid A of LPS isolated from rough (R)- or smooth (S)-form Gram-negative bacteria as well as to lipid A partial structures such as precursor IVA. To define the role of LBP in regulating responses to LPS we have examined TNF release in rabbit peritoneal exudate macrophages (M phi) stimulated with LPS or with complete or partial lipid A preparations in the presence or absence of LBP. In the presence of LBP, M phi showed increased sensitivity to S- and R-form LPS as well as synthetic lipid A. Compared with LPS or lipid A, up to 1000-fold greater concentrations of partial lipid A structures were required to induce TNF production. However, consistent with our previous observations that these structures bind to LBP, TNF production was increased in the presence of LBP. In contrast, LBP did not enhance or inhibit TNF production produced by heat-killed Staphylococcus aureus, peptidoglycan isolated from S. aureus cell walls, or PMA. Potentiated M phi responsiveness to LPS was observed with as little as 1 ng LBP/ml. Heat-denatured LBP (which no longer binds LPS), BPI (an homologous LPS-binding protein isolated from neutrophils), or other serum proteins were without effect. LBP-treated M phi also showed a more rapid induction of cytokine mRNA (TNF and IL-1 beta), higher steady-state mRNA levels and increased TNF mRNA stability. These data provide additional evidence that LBP is part of a highly specific recognition system controlling M phi responses to LPS. The effects of LBP are lipid A dependent and importantly, extend to LPS preparations isolated from bacteria of R- and S-form phenotype.     

Kinetics and mechanism of the oxidation of ammineruthenium(II) complexes by bromine

The reactions of Ru(NH₃)₅py²⁺, Ru(NH₃)₄bpy²⁺, Ru₂(NH₃)₁₀pz⁵⁺, RuRh(NH₃)₁₀pz⁵⁺ and Ru(NH₃)₅pz²⁺ with bromine are first-order in ruthenium and first-order in bromine. The rates decrease with increasing bromide ion concentration and, except for Ru(NH₃)₅pz²⁺, are independent of hydrogen ion concentration. The reactions are postulated to proceed via outer-sphere, one-electron transfer from Ru(II) to Br₂ with the formation of Br₂⁻ as a reactive intermediate. The bromide inhibition is ascribed to the formation of Br₃⁻ which is unreactive in outer-sphere reactions because of the barrier imposed by the need to undergo reductive cleavage. The reaction of Ru(NH₃)₅pz²⁺ is inhibited by hydrogen ions. The hydrogen ion dependence shows that Ru(NH₃)₅pzH³⁺ has a pK_a of 2.49 and is at least 500 times less reactive than Ru(NH₃)₅pz²⁺. The reaction of Ru₂(NH₃)₁₀pz⁴⁺ with bromine is biphasic. The second phase has a rate identical to that of the Ru₂(NH₃)₁₀pz⁵⁺-Br₂ reaction. A detailed analysis shows that the reaction of Ru₂(NH₃)₁₀pz⁴⁺ with bromine proceeds by a sequence of one-electron steps, Br₂⁻ being produced as an intermediate. A linear free energy relationship between rate constants and equilibrium constants, obeyed for all the reactions studied, provides an estimate of 1.5 × 10² M⁻¹ s⁻¹ for the self-exchange rate constant of the Br₂/Br₂⁻ couple.