Effect of Inflammatory Stimuli on the Silver Staining Pattern of the Rat Carotid Endothelium

Silver nitrate stains the intercellular junctions of the endothelium and other cytoplasmic or membrane components. Two protocols are described for the silver staining of rat carotid endothelium that exclude the use of pressurized fixatives and simplify the technique previously described for rat aorta. The entire surface of the carotid endothelium was examined and several parameters (stigmata, granularity, clustering of anionic sites, transversal lines, weakening of silver lines and leukocyte adhesion) were evaluated. We studied the pattern of silver staining in two situations: (1) endothelial activation and (2) neurogenic inflammation. Endothelial activation was produced by the intravenous administration of a proinflammatory albumin or polyinosinic acid. Both products cause a marked increase in leukocyte adhesion concomitant with a decrease in argyrophilia and a weakness or loss of silver lines. Neurogenic inflammation, which is mediated by substances released from sensory nerves, was induced by the intravenous administration of substance P or capsaicin. Both stimuli produced an increase in argyrophilia and weakness or loss of silver lines. Substance P caused a clustering of anionic sites, whereas this phenomenon was more discrete with capsaicin. Nearly 80% of all examined rats (controls and inflammatory stimuli treated) showed endothelial membrane disruptions formed by clusters of cells often in the shape of streaks aligned with the long axis of the vessel. The detection of these discontinuities is important, as loss of endothelial integrity is central in the initiation of pathological events.     

Dormancy in peach (Prunus persica L.) flower buds

Morphological studies were carried out with peach flower buds collected monthly in 1989 and 1990, from two months before leaf fall (7 March) until two to three weeks before bloom (7/8 August). Chilled (2–4°C for 30 days) and unchilled buds were exposed to 20 to 25°C, 100% RH and continuous light. Gibberellin A₃ (3 ng or 30 ng) was applied to some of the non-chilled cuttings at three days intervals. Then, 12, 19, and 26 days after they were planted, the buds were sampled and processed for histological studies. Cultured flower buds (chilled or unchilled) had accelerated anther and gynoecium morphogenesis after 12 days under controlled conditions, compared to buds processed immediately after collection from the field. Chilling treatment augmented the bud culture effect, while Gibberellin A₃ applications to the excised buds retarded bud morphogenesis to a stage comparable to that of buds collected directly from the field. This, suggests that the comparatively high levels of Gibberellin A_1/3 we previously found in mid winter [15, 18] could be at least one of the factors that controls floral bud dormancy by retarding anther and gynoecium development.     

Dynamic and steady state characteristics of the rabbit gastrocnemius muscle determined in series measurements

1. Within the range of the given conditions of measuring static and dynamic properties of the rabbit gastrocnemius muscle the following results were obtained: a) the dependence of the maxima of isotonic shortening upon the relative length of the muscle at constant load is linear; b) the parameters of the non-linear dependence of the passive elastic force of the muscle upon its relative length (measured in series) were identified using asymptotic regression; c) the time course of isotonic contractions (at an interval from 0 to 0.3 s after the beginning of stimulation) could be satisfactorily approximated by responses of a linear system to a step-function; d) the time course of isometric contractions (at an interval from 0 to 0.3 s after the beginning of stimulation) could be closely approximated by responses of a linear system to a step-function. 2. The time constants of isotonic and isometric contractions were determined as the parameters of the corresponding linear systems. 3. The maximum rates of the isometric and isotonic contractions were determined as maxima of the first derivatives of the responses of the corresponding models. 4. The experimental set-up made it possible to compare the values of the parameters concomitantly followed at various muscle lengths and at various loads.     

Synthesis and structure-activity relationship of 2-(aminoalkyl)-2,3,3a,8-tetrahydrodibenzo[c,f]isoxazolo[2,3-a]azepine derivatives: a novel series of 5-HT(2A/2C) receptor antagonists. Part 2.

Following the programme started at Janssen Research Foundation searching for 5-HT(2A/2C) antagonists, we now report on the synthesis of a series of substituted 2-(Dimethylaminomethyl)-2,3,3a,8-tetrahydrodibenzo[c,f]isoxazolo[2,3-a]azepine derivatives. The 5-HT(2A), 5-HT(2C) and H(1) receptor affinities as well as the mCPP antagonistic activity of the compounds synthesised is described.     

The effect of increasing glucose in CPD on the quality of stored blood

Changes in quality of blood units containing one and a half or double amounts of glucose, stored at +4 degrees C for three weeks were analysed. An experimental preservative containing glucose and fructose (1 : 1) was also used. No other additives (purine or purine-nucleoside) were applied. A standard CPD preservative of the National Inst. of Haematology and Blood Transfusion was used as control. The pH, plasma free haemoglobin, K+ content, red blood cell (RBC) ATP and 2,3-DPG content, and RBC fragility index were determined in each sample. Increase of glucose concentration, the addition of fructose had a beneficial effect on blood pH, and on plasma free haemoglobin and K+ concentration. 150% glucose improved the 2,3-DPG maintenance in stored blood.     

Identification of 5,6,7,8-tetrahydropterin and 5,6,7,8-tetrahydrobiopterin in Drosophila melanogaster

Using reversed-phase high-performance liquid chromatography with electrochemical detection we have demonstrated the occurrence of 5,6,7,8-tetrahydropterin and 5,6,7,8-tetrahydrobiopterin in Drosophila melanogaster. The former is the first time that has been detected in vivo. The identification has been based on the retention times, hydrodinamic voltagrams and the differential concentration in three strains of Drosophila melanogaster. Compared to the wild type, the Punch2 mutant has diminished levels of both pteridines, whereas Henna-recessive3 lacks completely tetrahydropterin and has increased levels of tetrahydrobiopterin, as expected according to their biochemical lesions.     

Effect of auto-oxidized phospholipids on oxidative enzyme assays based on tetrazolium salt reduction

The influence of auto-oxidized phospholipids on the reduction of the tetrazolium salt MTT coupled to the NAD+-dependent lactate dehydrogenase reaction was studied. The following results were obtained: (1) peroxidized phosphatidylcholine interfered in the time-course of the lactate dehydrogenase-mediated MTT reduction; (2) there was a time-dependent decrease in the hydroperoxide content of phosphatidylcholine vesicles during the incubation; (3) the diminution of phosphatidylcholine hydroperoxides required the presence of all the components of the system except MTT; (4) hydroperoxide diminution and MTT reduction were mediated by the superoxide radical O2-, since both processes were inhibited by superoxide dismutase; (5) EDTA inhibited the hydroperoxide decrease and abolished the interference of peroxidized phosphatidylcholine with MTT reduction. It was concluded that hydroperoxides compete with MTT for the electrons coming from substrate oxidation. The superoxide radical O2- and traces of some contaminating metal ion are involved in the process. This is a potential complication in the study of the effect of lipids on enzymatic activities assayed by the tetrazolium salt method.     

Hydrolysis of nucleic acids by sepharose-micrococcal endonuclease

Initial reaction rates for the hydrolysis of nucleic acids with micrococcal endonuclease (EC 3.1.31.1) insolubilized on Sepharose are strongly influenced by diffusional limitations. Although the absolute values are low, they can be increased substantially by changing particle and pore size of the support, or enzyme concentration in the insoluble derivative. As a result of steric and diffusional limitations, the course of the reaction and selectivity to hydrolysis products for the insoluble derivatives are different to those of the native enzyme; the former produces mainly large and small fragments but few of intermediate size. Because of these differences in course and selectivity of the reaction, diffusional limitations become less important when high initial reaction rates are not required.