Spin probe clustering in human erythrocyte ghosts

Summary A model has been developed for 5-nitroxide stearate, I(12,3), distribution in human erythrocyte ghosts which accurately predicts ESR spectral alterations observed with increased probe/total lipid (P/L) at 37°C. This spin probe occupies a class of high-affinity, noninteracting sites at low loading. Saturation occurs with increasing probe concentration, and, at higher loading, the probe inserts itself at initially dilute sites to form membranebound clusters of variable size. No low probe remains at high P/L where all I(12,3) clusters in a concentrated phase. This model allows determination of the dilute/clustered probe ratio, and shows that I(12,3) segregates in erythrocytes at what might otherwise be considered low P/L (e.g., 1/359). These findings validate the earlier use of empirical parameters to estimate probe sequestration in biological membranes.     

Studies on 9-amino-6-chloro-2-methoxy acridine fluorescence quenching and enhancement in relation to energy transduction in spheroplasts and intact cells of the cyanobacterium Plectonema boryanum

The fluorescent probe 9-amino-6-chloro-2-methoxy acridine was used to study the energy transduction in the thylakoid and cell membranes of the cyanobacterium Plectonema boryanum. Apart from light-driven electron transfer, the dark endogenous respiration also leads to energization resulting in an ACMA fluorescence response, that is sensitive to the electron flow inhibitor 2, 5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, to the energy transfer inhibitors dicyclohexylcarbodiimide and venturicidine and to the uncoupler 5-chloro-3-t-butyl-2-chloro-4-nitrosalicylanilide.In spheroplasts, in which the cell membranes have lost their capacity to maintain a proton gradient, the respiration-and light-induced ACMA fluorescence changes (quenching) are similar to those in chloroplasts. In intact cells a combination of reversible quenching and enhancement of ACMA fluorescence was found. This dualistic behaviour is supposedly caused by an opposite orientation of the thylakoid and cell membranes. ACMA quenching at the level of the thylakoids was obtained either by respiratory or photosynthetic electron transfer and gave similar responses to those obtained in the spheroplasts. The slower ACMA fluorescence enhancement, only observed in cells with intact cell membranes, also evoked by both respiration and light-induced energization is sensitive to the compounds mentioned above and in addition to KCN.Our results support the view [8] that dark oxidation of substrates by O₂ proceeds via the thylakoid membrane and terminates at a CN^- sensitive oxidase located in the cell membrane which requires the involvement of a mobile cytoplasmic redox mediator.Abbreviations ACMA 9-amino-6-chloro-2-methoxy acridine - chl a chlorophyll a - DBMIB 2, 5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCCD dicyclohexylcarbodiimide - DNP dinitrophenol - DNP-INT dinitrophenyl ether of 2-iodo-4-nitrothymol - FCCP carbonylcyanide-p-trifluoro-methoxy phenylhydrazone - S-13 5-chloro-3-t-butyl-2-chloro-4-nitrosalicylanilide - tricine N-2 (2-Hydroxy-1, 1-bis (hydroxymethyl) ethyl)-glycine - Tris Tris (hydroxymethyl) amino methane     

Insulin uptake by rat liver endothelium studied in fractionated liver cell suspensions

Summary Cellular distribution of insulin receptors was studied in fractionated rat liver cell suspensions using ¹²⁵1-insulin and a visual probe consisting of latex beads covalently linked to insulin (minibeads). Fractionation was done on metrizamide gradients which yielded two cellular fractions. The large cell fraction consisted mostly of hepatocytes and the small cell fraction consisted of 37% endothelial cells as well as Kupffer cells. The magnitude of insulin uptake by the endothelium-rich small cell fraction was at least double that of the uptake by the hepatocyte-rich fraction. The minibead technique demonstrated that in the small cell fraction only endothelial cells, and not Kupffer cells, were responsible for the insulin uptake. Our findings suggest that liver endothelium may be responsible for the uptake of circulating insulin and its transport to hepatocyte. This emphasizes the presence of a tissue-blood barrier in the liver.Abbreviations PRS phosphate-buffered saline - SEM scanning electron microscopy - TEM transmission electron microscopy     

Photochemical changes of fluorescent probes in membranes and their effect on the observed fluorescence anisotropy values

The fluorescence intensity of diphenylhexatriene (DPH) and of trimethylammonium-diphenylhexatriene (TMA-DPH) is measured when these probes are embedded in vesicles of dipalmitoyl- and dioleoylphosphatidylcholine (DPPC and DOPC), in mixtures of these vesicles as well as in vesicles of the mixed phospholipids, in trout intestinal brush border membranes and in mitoplasts of rat liver cells. The intensity in DOPC vesicles is found to be significantly higher than in DPPC vesicles. When these systems are irradiated with strong ultraviolet light radiation, a decrease in the fluorescence intensity is observed; this effect is much stronger in DOPC than in DPPC vesicles. The fluorescence anisotropy values in the mixture of vesicles as well as in the membranes show an initial increase with irradiation which is followed by a significant decrease. A transfer of DPH molecules between DPPC and DOPC vesicles is observed. For TMA-DPH this transfer takes place only from DPPC to DOPC vesicles, but not vice-versa. These results are related to intensity and anisotropy measurements of these probes in cell cultures.     

Sexing the human fetus and identification of polyploid nuclei by DNA-DNA in situ hybridisation in interphase nuclei

Samples of human adult lymphocytes, fetal lymphocytes, amniotic fluid cells, and chorionic villus cells were sexed independently by cytogenetics and DNA-DNA in situ hybridisation to a tritiated Y probe. For the in situ hybridisation analysis, the presence of Y bodies (hybridisation bodies) in 100 interphase nuclei were scored after autoradiography. In all, 82/83 samples were sexed in this way (one technical failure) and 78/82 were sexed by both in situ hybridisation and cytogenetics. There was complete agreement between the two methods. There was a considerable variation (40-100%) in the percentage of interphase nuclei with a hybridisation body among the male samples, but very few nuclei from female samples showed significant hybridisation. In situ hybridisation could be used to sex the conceptus when males but not females are at risk for various X-linked genetic disorders and may also be useful for detecting 45,X/46,XY mosaicism or polyploid/diploid mosaicism. This would be particularly useful for direct preparations of chorionic villus samples, which often prove difficult to analyse cytogenetically but offer the best means of avoiding maternal contamination. Some interphase nuclei had more than one hybridisation body, and this was most commonly found among amniotic fluid cells. Comparison of sizes of nuclei with one or two hybridisation bodies strongly suggested that most of the amniotic fluid cell nuclei with two hybridisation bodies were tetraploid.     

生物素标记寡核苷酸探针探测扩增的病理性突变珠蛋白基因

用末端转移酶催化生物素核苷酸底物(Biotin-ll-dUTP)共价连接在合成的寡核苷酸3’羟基末端,从而合成了两种寡核苷酸探针(β~T_(41-42)及β~A_(41-42))。用它们分别与克隆化扩增的正常和突变的β—珠蛋白基因片段杂变。结果表明该探针都具有与~(32)P探针相似的特异性,其杂交的灵敏度为2—3pg(特异序列)。进而将探测HbS基因的正常和异常两种寡核苷酸19聚体(β~A_6和β~S_6)用~(32)P和生物素分别标记;将HbS杂合子病人的白细胞DNA经聚合酶链反应(PCR)法扩增,并以含正常β—珠蛋白基因的DNA片段作对照,与两种探针分别进行斑点杂交。所得结果完全一致;Hbs杂合子DNA对正常和异常探针都显出杂交信号,而正常DNA只与β~A探针显杂交信号。     

A rapid method for the detection of potentially viable Mycobacterium leprae in human biopsies: a novel application of PCR

Abstract A simple procedure based on the polymerase chain reaction has been developed to detect Mycobacterium leprae , rapidly and unambiguously, in biological samples. Its application to small numbers of M. leprae cells (∼ 10²) isolated from armadillo liver, mouse footpads or human biopsies is discussed.     

Comparative measurements of the xylem pressure ofNicotiana plants by means of the pressure bomb and pressure probe

Determination of the pressure in the water-conducting vessels of intactNicotiana rustica L. plants showed that the pressure probe technique gave less-negative values than the Scholander-bomb method. Even though absolute values of the order of −0.1 MPa could be directly recorded in the xylem by means of the pressure probe, pressures between zero and atmospheric were also frequently found. The data obtained by the pressure probe for excised leaves showed that the Scholander bomb apparently did not read the actual tension in the xylem vessles ofNicotiana plants. The possibility that the pressure probe gave false readings was excluded by several experimental controls. In addition, cavitation and leaks either during the insertion of the microcapillary of the pressure probe, or else during the measurements were easily recognized when they occurred because of the sudden increase of the absolute xylem tension to that of water vapour or to atmospheric, respectively. Tension values of the same order could also be measured by means of the pressure probe in the xylem vessels of pieces of stem cut from leaves and roots under water and clamped at both ends. The magnitude of the absolute tension depended on the osmolarity of the bathing solution which was adjusted by addition of appropriate concentrations of polyethylene glycol. Partial and uniform pressurisation of plant tissues or organs, or of entire plants (by means of the Scholander bomb or of a hyperbaric chamber, respectively) and simultaneous recording of the xylem tension using the pressure probe showed that a 1∶1 response in xylem pressure only occurred under a few circumstances. A 1∶1 response required that the xylem vessels were in direct contact with an external water reservoir and/or that the tissue was (pre-)infiltrated with water. Corresponding pressure-probe measurements in isolated vascular bundles ofPlantago major L. orP. lanceolata L. plants attached to a Hepp-type osmometer indicated that the magnitude of the tension in the xylem vessels was determined by the external osmotic pressure of the reservoir. These and other experiments, as well as analysis of the data using classical thermodynamics, indicated that the turgor and the internal osmotic pressure of the accessory cells along the xylem vessels play an important role in the maintenance of a constant xylem tension. This conclusion is consistent with the cohesion theory. In agreement with the literature (P.E. Weatherley, 1976, Philos. Trans. R. Soc. London Ser. B23, 435–444; 1982, Encyclopedia of plant physiology, vol. 12B, 79-109), it was found that the tension in the xylem of intact plants under normal and elevated ambient pressure (as measured with the pressure probe) under quasi-stationary conditions was independent of the transpiration rate over a large range, indicating that the conductance of the flow path must be flow-dependent.     

Discrimination between closely related Triticeae species using genomic DNA as a probe

Summary Labelled total genomic DNA was used as a probe in combination with blocking DNA to discriminate between taxonomically closely related species in the genera Hordeum and Secale. Discrimination was possible both by Southern hybridization to size-fractionated restriction enzyme digests of genomic DNA and by in situ hybridization to chromosome preparations. To distinguish between two species (e.g. H. vulgare and H. bulbosum), genomic DNA from one species was used as the labelled probe, while unlabelled DNA from the other species was applied at a much higher concentration as a block. The blocking DNA presumably hybridized to sequences in common between the block and the labelled probe, and between the block and DNA sequences on the membrane or chromosomes in situ. If so, mainly species-specific sequences would remain as sites for probe hybridization. These species-specific sequences are dispersed and represent a substantial proportion of the genome (unlike many cloned, species-specific sequences). Consequently, rapid nonradioactive methods detected probe hybridization sites satisfactorily. The method was able to confirm the parentage of hybrid plants. It has potentially wide application in plant breeding for the detection of alien DNA transfer, and it can be easily adapted to many species.     

Sensitive detection of mycoplasmalike organisms in field-collected and in vitro propagated plants of Brassica, Hydrangea and Chrysanthemum by polymerase chain reaction

A polymerase chain reaction (PCR) protocol, previously designed for amplification of a DNA fragment from aster yellows mycoplasmalike organism (MLO), was employed to investigate the detection of MLO DNA in field-collected and in vitro micropropagated plants. PCR with template DNA extracted from symptomatic, naturally-infected samples of Brassica, Chrysanthemum and Hydrangea, each yielded a DNA band corresponding to 1.0 Kbp. However, no DNA product was observed when either infected Ranunculus (with phyllody disease) or Gladiolus with (symptoms of ‘germs fins’) was used as source of template nucleic acid for PCR; further experiments indicated absence of target DNA in the case of Ranunculus and the presence of substances in Gladiolus which inhibited the PCR. The MLO-specific DNA was detected by PCR using less than 95 pg of total nucleic acid (equivalent to total nucleic acid from 1.9, ug tissue) in the case of field-collected Hydrangea and less than 11.4 pg of nucleic acid (equivalent to total nucleic acid from 19 ng of tissue) in the case of field-collected Brassica. The findings illustrate highly sensitive detection of MLOs in both field-grown and in vitro micropropagated infected plants.