The application of laser microprobe mass analysis to the study of biological material

Laser microprobe mass analysis (LAMMA) is an investigational method which is a powerful tool for the identification and quantitation of various elements present in small volumes of tissue. LAMMA is highly sensitive and capable of rapidly detecting concentrations of 1–3 p.p.m. of most metallic elements, in precisely localized cellular compartments. In order to further assess its value, cultured skin fibroblasts and biopsy tissues from human subjects and experimental animals were probed by LAMMA, and the results were correlated with ultrastructural findings. Biopsy samples were obtained from patients suffering from Gaucher disease, and from patients and animals with pathologic iron or copper metabolism. No significant abnormalities were detected in the cultured fibroblasts from patients with Gaucher disease, in contrast to the iron content of tissue biopsy Gaucher cells, which was markedly increased, apparently as a consequence of erythrophagocytosis. Particularly intense iron-related peaks were found in liver cytosiderosis due to neonatal or genetic haemochromatosis, thalassaemia major and in animal models of iron overload. An additional finding was the presence of aluminium accumulation in siderosomes of different cells. In liver biopsy samples from human Wilson's disease and from rats with an inherited disorder causing copper toxicosis, copper-containing compounds were identified and localized, and their relative concentration was estimated by LAMMA. The present study showed that LAMMA is a valuable technique for the localization and estimation of relative abundance of trace elements in various tissues containing excessive amounts of metals.     

The use of sequence-specific antibodies to identify a secondary binding site in thrombin

The peptide comprising residues 62-73 of the B-chain of human alpha-thrombin was synthesized and polyclonal antibodies raised against it. These antibodies were found to bind to the synthetic peptide, a CNBr fragment, and a proteolytic subfragment containing this sequence, as well as the entire thrombin molecule. The purified antibodies had no effect on the hydrolysis by thrombin of D-Phe-pipecolyl-Arg-p-nitroanilide and caused only a minimal decrease (20%) in the second-order rate constant for inactivation by antithrombin III. On the other hand, the antibodies competitively inhibited the binding of hirudin over the concentration range tested (0-43 nM), and a dissociation constant of 3.4 +/- 0.5 nM was found for the antibodies. The release of fibrinopeptide A from the A alpha-chain of fibrinogen by thrombin was competitively inhibited with an inhibition constant of 11.7 +/- 0.4 nM. The activation of protein C by thrombin in the presence of thrombomodulin was also inhibited by the antibodies, and an apparent inhibition constant of 10.7 +/- 1.5 nM was found. In contrast, the antibodies had no effect on the activation of protein C in the absence of thrombomodulin. These results are discussed in relation to data obtained recently on the interaction of well defined proteolytic derivatives of human alpha-thrombin with the ligands described above.     

The solubility of calcium oxalate in tissue culture media

The equilibrium parameters for calcium oxalate solubility in tissue culture media were investigated because of the current interest in oxalate toxicity. The calcium selective ion electrode methodology was evaluated and calcium concentrations from potentiometric calculations were verified by d-c argon plasma emission spectroscopy. The experimental K(sp)'s at 25 degrees C for Dulbecco's modified Eagle media and McCoys 5A media are equivalent to the literature K(sp) of 2.3 x 10(-9) for low ionic strength. The equilibrium concentration products, [Ca2+] [C2O2-(4)], are ten times higher than the K(sp)'s due to the high ionic strengths of tissue culture media. At 37 degrees C, addition of soluble oxalate at the 10(-3) to 10(-4) M level causes >50% precipitation of the oxalate resulting in equilibrium oxalate concentrations of less than 6 x 10(-5) M. This relatively inexpensive selective ion technique allows the determination of oxalate concentrations in equilibrium-saturated media which are substantially less than those calculated by the amount of soluble oxalate added to the media.     

X-ray microprobe analysis of epithelial calcium transport

The sternal epithelium of Porcellio scaber was used as a novel model to study the subcellular elemental distribution in control and Ca(2+)-transporting stages in situ. The anterior sternal epithelium (ASE) is specialized for transport of cuticular Ca to sternal CaCO(3) deposits during premolt, and from these deposits during intramolt. The less specialized posterior sternal epithelium transports Ca(2+) to and from the cuticle. In the ASE cells basal [Na], [Cl], and [Mg] are higher than in the apical side. The basal [Na] increases from 105 to 173 mmol/kg dry mass between control and Ca(2+)-transporting stages, accompanied by a decrease in [Cl] and [K]. The [Mg] increases, suggesting transepithelial Mg(2+)-transport. Cytosolic [Ca] varied insignificantly between 4.5 and 5.7 mmol/kg dry mass, however, the number of Ca hot-spots with concentrations between 15 and 50 mmol/kg dry mass increased during transport. Mitochondrial [Ca] decreased in the ASE from 3.3 in the control to 1.0 in the late premolt and to 2.0 mmol/kg dry mass in the intramolt stage. The results suggest Na(+)-dependent mechanisms for transcellular Ca(2+)-transport and the presence of Ca(2+)-binding proteins. Organelles, probably the smooth endoplasmic reticulum, sequester Ca(2+) during intracellular Ca(2+)-transport. A role of mitochondria as a storage site for cuticular Ca is excluded.     

The correlation of individual results in laser microanalysis applied to histological slices

Summary This work is a continuation of investigations concerning the applicability of laser analysis in histochemical techniques (Kozik et al., 1970a, b; Kozik et al., 1971a, b).Agar standards containing known amounts of Pb were subjected to laser microanalysis and optical densities of the resulting Pb spectral bands as well as volumes of the resultant craters were determined.—Based on mathematical considerations and equation has been educed by means of which the experimentally obtained photometric readings may be converted into densities corresponding to mean crater volumes. In that way it is possible to eliminate inconveniences resulting from diversity of individual crater volumes and from eventual differences in the degree of atomic excitation between samples. Using this method, the concentrations of the investigated element in histological slices become expressed in percentages.     

Element analysis in fungal tissues using laser microprobe

Summary Fungal tissues were analysed for trace elements using emission spectroscopy with a laser. This method requires little sample preparation and has the advantage of not requiring large amounts of tissue. Laser microscopy permits the detection of 74 elements of the periodic table, from lithium (3) to uranium (92).     

Urinary glycosaminoglycans and glycoproteins in a calcium oxalate crystallization system

This study measures the effects of total urinary glycosaminoglycans (GAGs), glycoproteins (GPs) and individual GAGs on the nucleation rates (Bo), growth rates (G) and suspension densities (Mт) of calcium oxalate (CaOx) crystallization by the mixed suspension mixed product removal (MSMPR) system. Total urinary GAGs, glycoproteins and individual GAGs including heparan sulfate (HS), chondroitin sulfate (CS) and Hyaluronic acid (HA) were added into the artificial urine (AU) and then introduced into the MSMPR test chamber and the crystal sizes and numbers were analyzed by a particle counter. The effects of added GAGs and GPs on CaOx crystallization were reflected by the changes on the crystallization indexes including the Bo, G and Mт of CaOx that were calculated based on the crystal size and numbers. Total urinary GAGs showed no statistical significance on CaOx crystallization. However, individual GAGs such as HA, CS and HS enhanced Bo and suppressed the G when measured individually. CS and HS enhanced the Mт while HA shown no significant change in the Mт of CaOx. Total urinary GPs showed an increase in the G and Mт of crystals. Although total urinary GAGs showed no statistically significant effect on CaOx crystallization, individual GAGs (CS, HS) promoted the CaOx crystallization by increasing the suspension density of smaller crystals, indicative of reduced risk of stones while HA showed no significance in the M(T) of CaOx formed. Urinary GPs indicated increased sizes and M(T) suggesting larger crystals and/or aggregates.     

Calcium and oxalate content of the leaves of Phaseolus vulgaris at different calcium supply in relation to calcium oxalate crystal formation

Soluble and insoluble oxalate and insoluble calcium were measured in the leaves of Phaseolus vulgaris. The plants were grown in nutrient solutions with two different concentrations of calcium. Two developmental stages of the leaves were studied. Although the content of insoluble calcium differs widely according to leaf age and growth conditions, the percentage bound in crystals is nearly the same in all cases. In the growing leaves, concentrations of total oxalate are independent of calcium supply, thus, showing that the known rise in numbers of crystals, and of cells containing them, is not induced via oxalate biosynthesis. Fully expanded leaves contain more oxalate when grown in a nutrient solution with higher calcium concentration. Amounts of oxalate in percent of dry weight are similar to those given in the literature for other legume leaves.     

The use of whole-body sodium, potassium and calcium content to identify the nutrient status of first year salmon fry

Samples of fed and unfed hatchery reared Atlantic salmon fry and parr were analysed for body water and whole body Na, K and Ca content. From these body parameters it is possible to estimate the relative proportions of the skeletal mass, and intra- and extracellular spaces. These data can be applied to estimating the nutritional status of parr sampled at random from hatchery, and possibly from wild populations. When fish with a dry weight of about 25 and 70 mg were deprived of artificial food for 27 and 21 days, respectively, the Na/K ratio rose to 0.59 and 0.80 compared with 0.37 in the controls. With fish of about 475 mg in weight, percentage water content was a better indicator of undernourishment. The use of the K⁺: Ca²⁺ ratio to compare the amount of cellular material with the skeletal mass was a good indicator of nutritional depletion, especially in smaller fish.     

Engineering calcium oxalate crystal formation in Arabidopsis

Many plants accumulate crystals of calcium oxalate. Just how these crystals form remains unknown. To gain insight into the mechanisms regulating calcium oxalate crystal formation, a crystal engineering approach was initiated utilizing the non-crystal-accumulating plant, Arabidopsis. The success of this approach hinged on the ability to transform Arabidopsis genetically into a calcium oxalate crystal-accumulating plant. To accomplish this transformation, two oxalic acid biosynthetic genes, obcA and obcB, from the oxalate-secreting phytopathogen, Burkholderia glumae were inserted into the Arabidopsis genome. The co-expression of these two bacterial genes in Arabidopsis conferred the ability not only to produce a measurable amount of oxalate but also to form crystals of calcium oxalate. Biochemical and cellular studies of crystal accumulation in Arabidopsis revealed features that are similar to those observed in the cells of crystal-forming plants. Thus, it appears that at least some of the basic components that comprise the calcium oxalate crystal formation machinery are conserved even in non-crystal-accumulating plants.     

Cell and calcium oxalate crystal growth is coordinated to achieve high-capacity calcium regulation in plants

Summary Crystal idioblasts are cells which are specialized for accumulation of Ca²⁺ as a physiologically inactive, crystalline salt of oxalic acid. Using microautoradiographic, immunological, and ultrastructural techniques, the process of raphide crystal growth, and how crystal growth is coordinated with cell growth, was studied in idioblasts ofPistia stratiotes. Incorporation of⁴⁵Ca²⁺ directly demonstrated that, relative to surrounding mesophyll cells, crystal idioblasts act as high-capacity Ca²⁺ sinks, accumulating large amounts of Ca²⁺ within the vacuole as crystals. The pattern of addition of Ca²⁺ during crystal growth indicates a highly regulated process with bidirectional crystal growth. In very young idioblasts,⁴⁵Ca²⁺ is incorporated along the entire length of the needle-shaped raphide crystals, but as they mature incorporation only occurs at crystal tips in a bidirectional mode. At full maturity, the idioblast stops Ca²⁺ uptake, although the cells are still alive, demonstrating an ability to strictly regulate Ca transport processes at the plasma membrane. In situ hybridization for ribosomal RNA shows young idioblasts are extremely active cells, are more active than older idioblasts, and have higher general activity than surrounding mesophyll cells. Polarizing and scanning electron microscopy demonstrate that the crystal morphology changes as crystals develop and includes morphological polarity and an apparent nucleation point from which crystals grow bidirectionally. These results indicate a carefully regulated process of biomineralization in the vacuole. Finally, we show that the cytoskeleton is important in controlling the idioblast cell shape, but the regulation of crystal growth and morphology is under a different control mechanism.Abbreviation SEM scanning electron microscopy     

The use of slaughterhouse-obtained small intestinal tissue as control material in histological studies should be applied with prudence

This study aimed to evaluate the reliability of slaughterhouse-obtained small intestinal tissue as control material in equine colic research where molecular stress responses in small intestinal tissue are investigated. For this purpose, small intestinal samples from colic horses were collected during surgery or immediately after euthanasia at the oral border of strangulation resection sites and routinely processed for histopathology (i.c. rinsed with 4°C Krebs' solution, fixated overnight with 4% neutral buffered formaldehyde (FH) at room temperature). Control samples consisted of pieces of mid-jejunum, collected at the slaughterhouse and routinely processed for histopathology under 4 different conditions. The 4 conditions differed with regard to incubation and fixation temperature and whether or not oxygenated Krebs' solution was used. Histological scoring revealed that slaughterhouse samples had a higher mean lesion score (P<0.001) than colic samples. In addition, more slaughterhouse samples had a higher mean inflammation score than colic samples (P=0.001). The inflammatory cells in the small intestine consisted mostly of eosinophils and as such were very suggestive for parasitic infestation. Hypoxia-inducible factor-1α (HIF1α) nuclear immunoreactivity was more pronounced in slaughterhouse tissue, probably as a result of the delay between slaughter and sampling (P=0.034). The histopathological score (P=0.291), the inflammation score (P=0.248) and the HIF1α nuclear immunoreactivity (P=0.538) did not differ between the different collection protocols. It is concluded that slaughterhouse-obtained small intestinal tissue shows distinct alterations and that its use as control tissue when evaluating molecular stress responses should be applied with prudence.