Quantitative X-ray microanalysis of halogen elements in biological specimens

Summary Standards were prepared for quantitative X-ray microanalysis of the halogen elements Cl, Br and I in sections of resin embedded biological specimens. Halogenated aromatic compounds were dissolved in resin, which subsequently was polymerized. Homogeneity and stability of the standards were determined and found to be satisfactory. A general procedure of calculation of elemental concentrations according to the continuum method is given for the case that the specimen contains appreciable concentrations of one or more rather heavy elements. It is shown that use of approximations may lead to unacceptable errors, even in the concentration ranges occurring in biological specimens. As a practical application, the concentration of bromine in the chloroplasts of the red alga Chondrus crispus was determined quantitatively. The inner cells contained more bromine than the epidermal cells: in the chloroplasts of the inner cells bromine concentrations of about 6% could be demonstrated.     

Interaction of bromine with iodine in the rat thyroid gland at enhanced bromide intake

In experiments with rats, we have found that at enhanced intake of bromide, bromine does not replace chlorine in the thyroid; it replaces iodine. Under our experimental conditions, more than onethird of the iodine content in the thyroid was replaced by bromine. In the thyroid, bromine probably remained in the form of bromide and, in proportional to its increased concentration, the production of iodinated thyronines decreased, with the sum of the iodine and bromine concentrations being constant at the value of 20.51±1.16 μmol/g dry wt of the thyroid. In contrast to other organs, the biological behavior of bromine in the thyroid is not similar to the biological behavior of chlorine but resembles more that of iodine.     

Human biomonitoring of 73 elements in blood,serum, erythrocytes and urine

BackgroundHuman biomonitoring studies of trace elements in biological fluids are mostly limited to a certain number of elements or biological materials. In this study, we describe the significant extension of a biomonitoring to 73 elements being present in concentration ranges from ng/L to g/L in clinically relevant specimens such as blood, serum, erythrocytes and urine.MethodsThe samples were collected from 102 occupationally non-exposed inhabitants of northern Germany. The elements were determined either by inductively coupled plasma tandem mass spectrometry (ICP-MS/MS) in the low concentration range or by inductively coupled plasma optical emission spectrometry (ICP-OES) for essential trace elements and electrolytes.ResultsMean values and selected percentiles of element concentrations are presented for all sample materials. From the results, we calculated the distribution of elements between plasma and blood cells. Application of ICP-MS/MS improves selectivity and accuracy in the determination of elements that are strongly spectrally interfered, such as Cr, Ge, Pd or Ti in blood samples.ConclusionsThis publication provides very valuable information for occupational or environmental hygienists, toxicologists and clinical chemists due to the particularly high number of determined elements and presented concentration ranges.     

Two immunological approaches to the detection of ribulose-1,5-bisphosphate carboxylase in guard cell chloroplasts

Two immunological approaches were used to determine if ribulose bisphosphate carboxylase oxygenase (RuBisCo) is present in guard cell chloroplasts. Immunocytochemistry on thin plastic sections using tissue samples that were processed using traditional glutaraldehyde/osmium fixation and then restored to antigenicity with metaperiodate treatment, resulted in labeling over wild-type mesophyll and guard cell plastids of several green and white variegated Pelargonium chimeras. The density of immunogold labeling in guard cell chloroplasts was only about one-seventh of that noted in mesophyll chloroplasts on a square micron basis. Because guard cell chloroplasts are much smaller than mesophyll chloroplasts, and occur at lower quantities/cell, the relative differences in RuBisCo concentration between the cell types indicate that guard cells have only 0.48% of the RuBisCo of mesophyll cells. No reaction was noted over 70S ribosomeless plastids of these chimeras even though adjacent green chloroplasts were heavily stained, indicating the high specificity of the reaction for RuBisCo. Spurr's resin gave the most successful colloidal gold labeling in terms of low background staining and structural detail but L. R. White's resin appeared to be superior for antigen retention. In the white leaf edges of the white and green Pelargonium chimeras, the only green, functional chloroplasts are in the guard cells. When either whole tissue or plastid enriched extracts from this white tissue were electrophoresed, blotted, and probed with anti-RuBisCo a large subunit band was detected, identical to that in the green tissue. These data indicate that a low, but detectable, level of RuBisCo is present in guard cell chloroplasts.     

NaCl胁迫对宁夏枸杞叶和幼根显微及超微结构的影响

以宁夏枸杞为材料,采用超薄切片技术制备样品,应用光学显微镜和透射电镜分析了不同浓度NaCl胁迫条件下宁夏枸杞叶和幼根显微及超微结构的变化。结果表明：随着NaCl胁迫的加重,(1)叶片上表皮细胞增厚,栅栏组织细胞出现缩短现象,排列疏松且紊乱;幼根的初生结构无明显变化。(2)叶片栅栏组织中叶绿体不再紧靠在细胞膜上,叶绿体双层膜破坏,基粒片层松散排列,杂乱无章,出现膨胀和空泡现象,淀粉粒和嗜锇颗粒增多,叶肉细胞中线粒体发生轻微变化;幼根中皮层薄壁细胞线粒体形状发生改变,结构破坏,内膜和外膜模糊甚至破裂,大多数嵴模糊,出现空泡现象;细胞核解体,基质外溢。研究表明, 不同浓度的NaCl胁迫对宁夏枸杞叶片和幼根细胞的显微及超微结构影响不同,NaCl浓度大于200 mmol/L时,宁夏枸杞叶片和幼根细胞的显微及超微结构发生了明显变化,且叶肉细胞中线粒体的变化没有叶绿体的变化显著,推测叶肉细胞中线粒体的耐盐性比叶绿体强。     

Bromide kinetics and distribution in the rat. I

Biological half-lives of bromine in 15 different organs and tissues of the rat, in addition to the whole-body half-life, were determined by measuring the radioactive concentration of 82Br-bromide in samples of tissues collected at the time intervals of 12-396 h from animals that continuously (up to 17 d) received 82Br-labeled bromide in their drinking water. The half-life values, calculated from the experimental data by the method of gradual estimates of the parameters in question with the SPSS statistical program, ranged from 94.3+/-14.6 h in the thyroid gland to 235.0+/-88.9 h in liver. In most of the studied tissues, the biological half-lives of bromine were shorter than in the whole body, in which it equaled 197.8+/-22.2 h. Significant correlation between the values of the steady-state concentration of bromide and of the biological half-life was found for most tissues (except for liver). The steady-state concentrations of 82Br in tissues are probably proportional to the magnitude of bromide space, and, consequently, of chloride space.     

Sugar compartmentation in frost-hardy and partially dehardened cabbage leaf cells

In frost-hardy and partially dehardened leaves of Brassica oleracea L. var. sabellica L. the distribution of cryoprotective sugars and of chloride between chloroplasts and the nonchloroplast part of leaf cells was investigated using the nonaqueous isolation technique as a means of cell fractionation. In chloroplasts of frost-hardy leaves high concentrations of sucrose and raffinose and comparatively low concentrations of chloride have been found. The ratios between sugars and chloride were so as to ascertain complete protection of the frost-sensitive thylakoid membranes during freezing. During dehardening, sugars decreased especially in the chloroplasts. There was a conversion of sucrose and raffinose into monosaccharides. This led to a large increase in the concentration of glucose and fructose in the nonchloroplast parts of the cells. There is evidence that the sugar concentration in the vacuole increased at the expense of sugars located in chloroplasts and cytoplasm. The quantity of sugars that remained in the chloroplasts did not appear to be sufficient for complete membrane protection at very low freezing temperatures.     

Secondary ion mass spectrometry (SIMS) of standards for analysis of soft biological tissue.

Gelatin films containing water-soluble salts of lithium, rubidium, strontium, or copper were analyzed by secondary ion mass spectrometry. Calcium and vanadium organometallic compounds in an epoxy resin were similarly analyzed. A linear relationship between positive secondary ion intensity and ion concentration was observed over several decades of ion concentration and at absolute concentrations as low as 1 wt ppm. These standards can be used for quantitative analysis of tissue or other biological material in epoxy resins, providing a highly sensitive method for simultaneous quantitation and localization of elements.     

中国树花对乌鲁木齐南郊空气污染生物指示的研究

目的：中国树花对乌鲁木齐南郊空气污染生物指示作用的探讨。方法：采用电感耦合等离子光谱直读法测定移植地衣内重金属含量的变化。结果：地衣体内被测定出的重金属元素种类和含量与移植时间的长短之间存在显著性差异。对照组中,中国树花体内仅测出2种元素,其中Fe和Mn的含量分别为1.2915±0.054μg.g-1和0.0261±0.001μg.g-1。移植1个月后测出Al、Fe、Mn、Zn等4种元素,其含量分别为0.68±0.036、0.706±0.086、0.021±0.016和0.061±0.005μg.g-1;移植3个月后测出Al、Cr、Cu、Fe、Mn、Ni、Zn等7种重金属,其含量分别为0.285±0.039、0.0032±0.001、0.004±0.002、0.499±0.133、0.106±0.015、0.011±0.01、0.011±0.01、0.038±0.002μg.g-1。结论：中国树花在生物评价乌鲁木齐南郊空气污染方面具有一定的研究价值。     

Glucose transport into spinach chloroplasts

The uptake of radioactively labeled hexoses and pentoses into the sorbitol-impermeable (3)H(2)O space (the space surrounded by the inner envelope membrane) of spinach (Spinacia oleracea L.) chloroplasts has been studied using silicone layer filtering centrifugation. Of the compounds tested, d-xylose, d-mannose, l-arabinose, and d-glucose are transported most rapidly, followed by d-fructose and l-arabinose. The rate of l-glucose uptake is only about 5% of that of d-glucose.The transport of d-glucose and d-fructose shows saturation characteristics, the K(m) for d-glucose was found to be about 20 mm. All sugars transport and phloretin inhibit d-glucose transport. The temperature dependency of d-glucose transport appears to have an activation energy of 17 kcal/mol.With low external concentrations of d-glucose the transport into the chloroplasts proceeds until nearly the external concentration is reached inside the chloroplasts.d-glucose transport is inhibited by high d-glucose concentrations in the medium. It is concluded that d-glucose and other hexoses are transported by carrier-mediated diffusion across the inner envelope membrane. This transport is similar to the transport of d-glucose into erythrocytes.     

BROMINE IN THE CUTICLE OF POLYSIPHONIA NIGRESCENS: LOCALIZATION AND CONTENT1

The cuticles and chloroplasts of young and old fronds of Polysiphonia nigrescens (Huds.) Grev. were analysed by quantitative X-ray microanalysis for their content of bromine. Cuticles of old fronds contained 1.7 ± 0.14% bromine while those of young ones contained only 0.94 ± 0.22% bromine. The bromine content of the chloroplasts showed considerable variations between individual chloroplasts, but was found not to increase with age. Young fronds contained four separate layers in the cuticle when observed under the electron microscope. In the old fronds three of these layers disappeared while the remaining one became thick and very electron dense.     

Immunogold localization of photosynthetic enzymes in leaves of Aristida latifolia, a unique C4 grass with a double chlorenchymatous bundle sheath

C₄ species of the genus Aristida (Poaceae) have 3 distinct types of photosynthetic cells in their leaves, the mesophyll (M) cells, the outer bundle sheath (BS) cells, and the inner BS cells, and exhibit a unique Kranz-type leaf anatomy. The cellular localization of C₃ and C₄ photosynthetic enzymes was investigated in leaves of Aristida latifolia Domin by the protein A-gold immunocytochemical technique. The outer BS cells contained centripetally located small chloroplasts, which were structurally similar to those of the M cells. The inner BS cells contained centrifugally located large chloroplasts, which lacked well-developed grana and exhibited rudimentary grana for the most part. The leaves contained high levels of NADP-malic enzyme (EC 1.1.1.40) activity, and the plant was classified as being of the NADP-malic enzyme type. The immunocytochemical study revealed that labeling of ribulose 1,5-bisphos-phate carboxylase/oxygenase (EC 4.1.1.39) was present in the chloroplasts of the outer and inner BS cells, but was undetectable in the M cells. Labeling of phoshoen-olpyruvate carboxylase (EC 4.1.1.31) was observed in the cytosol of M cells, but not in that of BS cells. By contrast, labeling of pyruvate, P_i dikinase (PPDK, EC 2.7.9.1) was evident not only in the chloroplasts of M cells but also in those of outer BS cells, but was absent from the inner BS cells. The density of labeling in the chloroplasts of M cells was higher than that in chloroplasts of outer BS cells. These results indicate that the two carboxylating enzymes are differentially distributed between the M cells and the two types of BS cells, whereas PPDK shows a more complex distribution pattern. The locations of these enzymes are discussed in relation to C₄ photosynthesis.     

Reversible swelling and contraction of isolated spinach chloroplasts

By use of a micro technique for producing extracts of spinach mesophyll cells, chloroplasts were isolated in a state wherein they displayed microscopically visible, reversible osmotic properties. Swollen spherical chloroplasts treated with hypertonic sucrose or mannitol media, but not NaCl, could be shrunken to a state resembling their disk appearance in living cells. Reversible osmotic behavior was more easily demonstrated when the chloroplasts were initially isolated from cells in a relatively low osmolar concentration in contrast to using 0.25 m sucrose or more concentrated media. Individual chloroplasts could be swollen and contracted repeatedly through as many as 4 cycles. The relationship between the capacity for osmotic behavior and chloroplast appearance in cell extracts is discussed.     

Identification of the photosynthetic CO2 fixation inhibitors in isolated pine chloroplasts as resin acids

Thus far all attempts to isolate CO, fixing chloroplasts from pine have failed. In this paper it is proposed that resin acids present in pine needles partition into membranes during chloroplast isolation and interfere with specific reactions of the Calvin cycle. CO, fixation by isolated spinach chloroplasts was strongly inhibited by the introduction of a suspension of chloroplasts isolated from Pinus sylvestris L. A partially purified organic extract obtained from chloroplasts of this pine species also strongly inhibited CO, fixation by the spinach chloroplasts. The major inhibitory compounds from the organic extract were identified as a mixture of resin acids by gas-liquid chromatography and mass spectrometry. Two resin acids, abietic acid and dehydroabietic acid, were tested for inhibitory activity. Both resin acids were potent inhibitors of photosynthetic CO₂fixation, with dehydroabietic acid being about three times more potent than abietic acid.     

Accumulation of bicarbonate in intact chloroplasts following a pH gradient

With silicone layer filtering centrifugation the uptake of radioactively labelled bicarbonate into isolated spinach chloroplasts was followed. This uptake was shown to have the following properties:

1. (a) It is so rapid that the kinetics of uptake usually cannot be resolved.

2. (b) Bicarbonate is accumulated in the stroma. The factor between the internal and external concentrations increases greatly when the pH of the medium is lowered from pH 8.5 to pH 7.0.

3. (c) The accumulation factor is independent of the concentration in the medium for a long concentration range.

4. (d) The accumulation of bicarbonate is increased when the chloroplasts are illuminated. This increase is abolished by the addition of uncoupler.

5. (e) Diamox, an inhibitor of carbonic anhydrase, inhibits the rate of bicarbonate uptake.

The activity of carbonic anhydrase was assayed in isolated chloroplasts and in leaf homogenates. In agreement with earlier reports the main activity was found to be located in the chloroplasts. This activity is latent; it can be only assayed if the chloroplasts are osmotically shocked.

From these results the following conclusions have been drawn:

1. (a) The inner membrane is impermeable to protons. Light-driven proton transport into the thylakoid space causes an alkalisation of the stroma.

2. (b) The uptake of bicarbonate proceeds via diffusion of CO₂ across the inner membrane. There are no indications for a specific transport of bicarbonate.

3. (c) The CO₂ concentration in the chloroplasts may be equal to the CO₂ concentration in the external space. The distribution of bicarbonate between the two compartments is inversely proportional to the distribution of protons.

A possible involvement of carbonic anhydrase and the bicarbonate pool in the stroma in increasing the CO₂ affinity of CO₂ fixation is discussed.     

Direct measurement of femtogram amounts of DNA in cells and chloroplasts by quantitative microspectrofluorometry

Absolute DNA amounts of individual chloroplasts were determined by measuring the fluorescence intensity of chloroplasts stained with 4',6-diamidino-2-phenylindole (DAPI) relative to that of the bacterium Pediococcus damnosus (cerevisiae) smeared on the same slide. An absolute DNA content of 7.7 X 10(15) g for a standard P. damnosus cell type was calculated by comparing the relative fluorescence values and frequency of each stage of cellular development in a culture to the average DNA content of all cell types determined by chemical methods. Chlorophyll was extracted from the chloroplasts during fixation so that chlorophyll autofluorescence was not present when DAPI fluorescence was measured. Absolute amounts of DNA could then be determined for single chloroplasts, either within cells that were individually selected from a mixed cell population or in small preparations of isolated chloroplasts. The DNA amounts of chloroplasts from mesophyll cells determined in this way were similar to the values previously determined by bulk averaging methods. Chloroplast DNA amounts from different cell types of the leaf could be measured by microspectrofluorometry, and it was found that chloroplasts from spinach epidermal cells contained about half as much DNA as chloroplasts from adjacent mesophyll cells.     

Determination of the lowest concentrations of aldehyde fixatives for completely fixing various cellular structures by real-time imaging and quantification

The effectiveness of fixatives for fixing biological specimens has long been widely investigated. However, the lowest concentrations of fixatives needed to completely fix whole cells or various cellular structures remain unclear. Using real-time imaging and quantification, we determined the lowest concentrations of glutaraldehyde (0.001–0.005, ~0.005, 0.01–005, 0.01–005, and 0.01–0.1 %) and formaldehyde/paraformaldehyde (0.01–0.05, ~0.05, 0.5–1, 1–1.5, and 0.5–1 %) required to completely fix focal adhesions, cell-surface particles, stress fibers, the cell cortex, and the inner structures of human umbilical vein endothelial cells within 20 min. With prolonged fixation times (>20 min), the concentration of fixative required to completely fix these structures will shift to even lower values. These data may help us understand and optimize fixation protocols and understand the potential effects of the small quantities of endogenously generated aldehydes in human cells. We also determined the lowest concentration of glutaraldehyde (0.5 %) and formaldehyde/paraformaldehyde (2 %) required to induce cell blebbing. We found that the average number and size of the fixation-induced blebs per cell were dependent on both fixative concentration and cell spread area, but were independent of temperature. These data provide important information for understanding cell blebbing, and may help optimize the vesiculation-based technique used to isolate plasma membrane by suggesting ways of controlling the number or size of fixation-induced cell blebs.     

Ultrastructure of chloroplasts in'Marimo'(Cladophora aegagropila, Chlorophyta), and changes after exposure to light

This study compares the ultrastructure of the inner, dark-habituated cells of the green ‘Cladophora-ball’, or Marimo, to that of similar cells at the surface. Cells not exposed to light possess fewer, but larger and more irregular, chloroplasts than do the outer cells. Unexposed chloroplasts have a pyrenoid matrix lacking starch sheaths and containing unusually thick granal stacks. Despite prolonged exposure to darkness, the chloroplasts contain small starch grains. After exposure to light, such chloroplasts divide, become smaller and take on the appearance of those in the outer layer cells. Within 48 h, all of the chloroplasts develop substantial starch grains and the pyrenoids are surrounded by starch sheaths. This response is consistent with previous reports of the recovery of photosynthetic activity in inner cells exposed to light.     

Chloride and sulphur concentrations in chloroplasts of spinach

Chloride concentrations within individual chloroplasts, the adjacent cytoplasm and nearby vacuoles of spinach mesophyll cells (Spinacea oleracea L. cv. Hybrid 102) were determined by means of electron probe X-ray microanalysis in the cleavage plane of quench frozen tissue, which was maintained at liquid nitrogen temperatures. The accuracy of quantitative data obtained with this technique is greatly improved by the adoption of a peak to background ratio method and use of carbon slurry standards, which mimic the quench frozen tissue and its X-ray fluorescence. Chloroplasts were incapable of maintaining relatively high levels of Cl^? under conditions of low Cl^? availability (zero Cl^? or 20 μM Cl^? in nutrient solution), and under conditions of Cl^? stress (100 or 200 mM Cl^?) chloroplasts had only a limited capacity to maintain a Cl^? concentration at a level below that of the cytoplasm and vacuole. However, under conditions of Cl^? stress the concentration of Cl^? in cytoplasm immediately adjacent to chloroplasts was substantially higher than in the chloroplasts or more distant cytoplasm. Thus, Cl^? levels in chloroplasts are apparently not as tightly regulated as was suggested by estimates of Cl^? concentration based on aqueous isolation of chloroplasts. Levels of S in chloroplasts were relatively high (equivalent to 40–60 mM SO₄^2? in S standards) and constant for all treatments, with the possible exception of lower S levels in chloroplasts of leaves approaching premature senescence as a result of salt stress. It is implied that the stability of the S-content results largely from its presence in macromolecular components of chloroplasts (sulfolipids and proteins).     

Trace element content in fingernails and hair of a nonindustrialized US control population

The concentrations of 17 elements in the nail and hair of 117 subjects from a nonindustrialized environment were determined by instrumental neutron activation analysis (INAA). A new method of statistical treatment that allows for more meaningful use of detection limit values was used to process the concentration data. Geometric means and standard errors are presented for each element, along with a summary of the effects of age, sex, and treatment on the concentration of each element. For nails, these data represent the first comprehensive study for several important elements. Correlations for each element between hair and nail were determined. With few exceptions, concentrations of nonessential trace elements were positively correlated in hair and nail, whereas concentrations of essential elements showed no correlations. The factors affecting concentrations and control levels must be considered in studying alterations in disease states.