Heat Stable Nuclease in Mastitic Milk

The production of a heat stable nuclease (deoxyribonuclease) by Staphylococcus aureus was first reported by Cunningham et al. (1956). The detection of the enzyme was simplified by Lachica et al. (1971), who developed an agar diffusion test based on the metachromatic properties of toluidine blue.     

Direct recording of metachromatic spectra in a model system of polyacrylamide films

Synopsis The metachromatic staining of polyacrylamide films containing different glycosaminoglycans is described. This model system made direct recording of metachromatic curves possible, under circumstances comparable to those of stained sections under the microscope, with virtually no interference of the corresponding orthochromatic peaks.The staining was carried out under standardized conditions (of buffer concentration, pH and temperature) and was shown to follow the Lambert-Beer law. The metachromatic peaks obtained with this system are listed for seven basic dyes, each complexed with seven different glycosaminoglycans.     

Localization of carboxyl groups in gastric mucosa as possible sites of gastrin

Summary Human and pig gastrins contain a sequence of five consecutive glutamic acid residues. An attempt was made to localize gastrin using methods known or assumed to operate on a carboxyl mechanism. General methods for acidic groups were combined with selective blocking (methylation) and unblocking (saponification) methods to increase COOH specificity. Epithelial cells with weakly metachromatic granules could be identified in untreated sections stained with toluidine blue (pH 5). After prolonged methylation and saponification, the same and previously obscured cells were moderately to intensely metachromatic, this residual basophilia attributable to weak COOH groups. Specifically marked metachromatic cells were iron-positive after colloidal iron staining, but were delineated easily only after methylation-saponification. Metachromatic cells were also clearly demonstrated by the carboxyl method of Barrnett and Seligman and by silver impregnation (pH 5). The granular metachromatic cell demonstrated by these methods contains significant amounts of a weakly acidic component which the Barrnett-Seligman reaction indicates to be glutamic acid. Comparable staining results were obtained with gastrin producing Zollinger-Ellison islet cell adenomas. It is postulated that the COOH-rich substance is gastrin or gastrin precursor and that the metachromatic cell is responsible for its production.Supported by General Research Support Grant No 5 S01 FR05411-06.     

A microfluorometric study of masked metachromasia in cells of the endocrine polypeptide (APUD) series

Synopsis Masked metachromasia can be demonstrated by staining with a metachromatic basis dye after a Feulgen-type hydrolysis of suitably fixed tissue, and is believed to be indicative of polypeptides with a high concentration of side-chain acidic groups and a random-coil conformation. In this investigation, the metachromatic fluorochrome Coriphosphine O was used. After staining, the degree of metachromasia under various conditions and in several tissues was assessed by microspectrofluorometric measurements of the ratio of metachromatic fluorescence (red) to orthochromatic fluorescence (green). This technique was employed, in the first instance, to determine the optimum staining conditions; details of the final staining method are presented. Measurements of metachromasia in different tissues under standardized conditions showed that the degree of metachromasia varied between different cell types in the APUD series.     

An Evaluation of the Metachromasia of Bromophenol Blue

The heterogeneity of bromophenol blue from different commercial sources was revealed by paper chromatography. Isopropanol:ammonia:water (20:1:2) as the solvent system gave the best separation. A variety of impurities: violet, pink, light blue and yellow coloured ones were observed. Two of the yellow fractions showed a spectral shift to red in the presence of ammonia vapour. The respone of the main dye component with the anionic chromotropes such as heparin and hyaluronate was found to be metachromatic similar to that exhibited by the dye solution and not due to a polychromatic effect. The metachromatic effect was blocked by FeCl₃ as in the case of cationic dye metachromasy. The observed metachromatic colour is not one of the colours which characterize those resulting from changes caused by pH.     

Differentiation of polyphosphate and poly-β-hydroxybutyrate granules in an Acinetobacter sp. isolated from activated sludge

Cells containing polyphosphate 71 micrograms P (mg protein)-1 and no poly-beta-hydroxybutyrate showed metachromatic granules but no lipid granules; cells containing poly-beta-hydroxybutyrate (15% of dry weight) showed fluorescence lipid granules but no metachromatic granules; whereas cells containing both polyphosphate and poly-beta-hydroxybutyrate showed both types of granules. These observations, together with a critical review of the literature, show a clear distinction between metachromatic (or volutin) granules and lipid granules.     

Accumulation of lysosulfatide (sulfogalactosylsphingosine) in tissues of a boy with metachromatic leukodystrophy

Abnormal accumulation of lysosulfatide (sulfogalactosylsphingosine) was evident in autopsied tissues from a boy with late-infantile metachromatic leukodystrophy. The concentration was high in the cerebral white matter, spinal cord and sciatic nerve (116-787 pmol/mg protein) and low in the cerebral gray matter, kidney and liver (4-40 pmol/mg protein). As is the case with galactosylsphingosine, lysosulfatide inhibited cytochrome c oxidase activity, in a dose-dependent manner. Judging from the tissue distribution of the accumulated lysosulfatide and because of the cytotoxicity, the lysosulfatide presumably explains the demyelination seen in the nervous tissues of patients with metachromatic leukodystrophy.     

An Evaluation of the Metachromasy of Anionic Dyes. II. Visual and Spectral Observations on Solutions

The reactions of 13 anionic dyes in solution with a basic protein (protamine), a cationic detergent, guanidine, histamine, procaine, quinine, and strychnine were examined visually and spectrophotometrically in order to distinguish metachromatic changes of the dyes. Disazo dyes (Congo red, benzopurpurin, but not trypan blue) were metachromatic; indigoid, triphenylmethane and xanthene dyes were not. The magnitude of metachromasy in this series of dyes was not great compared with cationic dyes, the shifts of absorbance maxima being only about 15 mμ against 90 mμ or more for some cationic metachromatic dyes. The most effective chromotropes were protamine and a cationic detergent. Agreement between visual observations on tissue sections, visual observations on solutions, and spectral observations on solutions was generally good.     

Histological, Chromatographic and Spectrophotometric Studies of Toluidine Blue

Chromatographic analysis of commercial batches of toluidine blue shows these to be dye mixtures. Histologically, some samples were found to be poor metachromatic dyes. These unsatisfactory stains contained blue dyes with little or no metachromatic properties as well as a metachromatic fraction. On the other hand, contaminating dyes in histologically satisfactory samples had poor staining qualities and hence did not interfere with the color produced by the metachromatic fraction.

Chromatographic fractionation of different commercial batches of toluidine blue yielded identical, homogeneous metachromatic dyes. These purified dyes had a peak absorption at 615 mμ in contrast to that of purified azure A whose peak absorption was at 622.5 mμ.     

Effects of Ethanol on the Metachromatic Reaction of Toluidine Blue O

Ethanol abolishes the metachromatic reaction of toluidine blue O with un-combined chromotropes but not when they are in association with protein. The green colour obtained in metachromatic regions is established as not due to any green impurity of the dye by chromatographic analysis but due to the fluid dehydrants combining with the dye as dye-organic solvent mixture showed green. The loss of metachromasia is not due to a dehydration effect of ethanol alone for the following reasons: (i) Stained samples of chromotropes dried in vacuuo continued to retain the metachromatic colour, (ii) Although other dehydrating agents likewise abolished the metachromasia, alcohols which have very slight affinity to water also abolished it, (iii) Ethanol does not abolish metachromasia produced in an acid mucopolysaccharide-protein complex. This has been suggested as due to the inability of ethanol to separate the dye from such compounds and to bring about a shift to green.     

Ultrastructural chemistry of isolated molecules. Application to biological macromolecules. Study of metachromasia

The ultrastructural chemistry on isolated macromolecules enables to localize chemical function on spread molecules by means of reactions whose product is visible in electron microscopy (metal colloids, lectins, etc.). The reaction of linear polyanions with metachromatic dyes (bifunctional ligands) results in the formation of metachromatic complexes with a periodic structure : ligand molecules are intercalated between the polyanions arranged in parallel arrays; this structure is correlated with the metachromasy.     

A 9-bp deletion (2320del9) on the background of the arylsulfatase A pseudodeficiency allele in a metachromatic leukodystrophy patient and in a patient with nonprogressive neurological symptoms

A 9-bp deletion (2320del9) was detected in the arylsulfatase A genes of a patient with late infantile metachromatic leukodystrophy and of a patient with nonprogressive neurological symptoms and very low arylsulfatase A activity. Both patients are heterozygous for the deletion, which involves codons 406–408 and causes loss of a Ser-Asp-Thr tract in the predicted protein. In both patients the 9-bp deletion lies in a pseudodeficiency allele. The patient with metachromatic leukodystrophy carries the common 459 + 1G > A mutation in the other allele. The other patient is homozygous for the pseudodeficiency allele, and consequently is a compound heterozygote for a metachromatic leukodystrophy allele and a pseudodeficiency allele. We hypothesize that the compound heterozygosity predisposes to the development of nonprogressive neurological symptoms in the presence of additional, still unknown, genetic or nongenetic factors. Received: 18 April 1997 / Accepted: 16 August 1997     

The Nucleolus and Parachromatin of the Ascites Tumor Cell

1. A method is described for distinguishing the ribonucleoproteins of the nucleolus and parachromatin of ascitic tumor cells of the mouse. 2. In these cells the transfer of ribonucleoprotein from the nucleus to the cytoplasm can occur in two ways. (a) At the end of prophase the nucleolus separates from the chromosomes and nucleolar fragments are released into the cytoplasm. (b) During prophase the parachromatin is aggregated to form parachromatin bodies which are discharged into the cytoplasm, where they can be detected during metaphase, anaphase, and telophase. 3. A metachromatic form of RNA is demonstrable, and may be synthesized, in close relation to the chromosomes during prophase, metaphase, and anaphase. During telophase the distribution of metachromatic RNA changes, the chromatin loses its metachromasia, and intranuclear metachromatic parachromatin becomes evident.     

Sulfatide excreting heterozygous carrier of juvenile metachromatic leukodystrophy or asymptomatic patient of adult metachromatic leukodystrophy.

In a family with juvenile metachromatic leukodystrophy (sulfatide lipidosis) 2 patients showed residual arysulfatase A activities of 5--6%. The patients' healthy father was characterized biochemically by a 39% normal activity of leukocyte plus plasma arylsulfatase A. The father was further characterized by a high sulfatide excretion (0.2--0.5 mg/I urine) and, paradoxically, by a normal sulfatide degrading enzyme activity in vitro. This special carrier is suspected to be heterozygous for a) arylsulfatase A deficiency and b) arylsulfatase A (sulfatidase) lability. This presumed additional genetic defect could be the cause of the sulfatide excretion which, in turn, would be a sign of the preclinical stage of an exceptional form of adult metachromatic leukodystrophy. The normal sulfatidase activity seems to be due to an in vitro effect.     

Histochemical conditions influencing metachromatic staining. A comparative study by means of a model system of polyacrylamide films

Synopsis The influence of different histochemical conditions on some metachromatic staining reactions has been studied using polyacrylamide films containing pure glycosaminoglycans. The films were incubated in fixatives without staining, and in glycerol, diethylene glycol and other glycols, formamide,N,N-dimethylformamide, dimethyl sulphoxide and ethanol (of several concentrations) after staining and their absorption (metachromatic) spectra recorded. In the case of heparin and heparan sulphate the metachromasy was disturbed when the films were immersed before staining in some fixative solutions containing formaldehyde and acid. After equilibration of stained films in organic solvents, changes in the absorption peaks were found to depend on the type and concentration of solvent, the type of glycosaminoglycan and the type of dye.Films containing glycosaminoglycan plus protein were used to investigate the blocking of the metachromatic reaction as the result of ionic interactions with proteins. The parameters that influence this phenomenon (e.g type of protein, glycosaminoglycan and dye, pH of staining) are discussed and a three-dimensional picture is introduced which can explain some of the results obtained in these experiments.     

Metachromatic staining and electron dense reaction of glycosaminoglycans by means of Cuprolinic Blue

Summary The cationic phthalocyanin-like dye Cuprolinic Blue, unlike phthalocyanin dyes such as Alcian Blue or Astra Blue, can definitely exhibit a clear metachromatic reaction with appropriate substrates, The application of Cuprolinic Blue to epoxy-embedded semithin sections revealed that mast cell cytoplasmic granules, goblet cell mucin and cartilage matrix stained in violet shades (metachromatic), whereas nuclear chromatin presented a bright blue coloration (orthochromatic). The metachromatic structures showed a high degree of contrast when ultrathin sections treated with Cuprolinic Blue were examined by electron microscopy.Cytophotometric measurements of stained components from the large intestine showed different absorption maxima: at 580 nm for mucin and at 640 nm for nuclei. The spectroscopical analysis revealed a clear-cut metachromatic shift when the dye was in the presence of chondroitin—4-sulphate. The addition of aluminium metal to Cuprolinic Blue solutions resulted in a striking spectral change; under such conditions the dye showed absorption maximum at 530 nm.     

Occurrence of the periodic acid-Schiff positive granular leucocyte (PAS-GL) in some fishes and its significance

Preparations of peripheral blood of 58 species of fish, mostly teleosts, were examined for the presence of granulocytes. Heterophils are commonly found but eosinophils are rare and metachromatic basophils never seen. Granulocytes (PAS-GL) with periodic acid-Schiff positive granules, were observed in 30 species representing marine and freshwater, and 'primitive' and 'advanced' types. It is postulated that the PAS-GL is the piscine forerunner to the heparin and histamine-containing metachromatic basophil/mast cell of higher vertebrates.     

Spectroscopic and thermodynamic investigations on the binding of azure B to chondroitin sulfate and the structure of the metachromatic dye complex

The binding of azur B to chondroitin sulfate (CHS) was investigated using absorption spectroscopy. In aqueous solutions it is possible to distinguish three different dye species with absorption bands at 646, 597, and 555 nm. They are assigned to monomers, dimers, and higher aggregates of azure B, which become bound to CHS as the dye concentration (CD) increases. The short-wavelength band (555 nm) causes metachromasia in stained histological materials. When saturation occurs, the metachromatic azure B-CHS complex has a 1:1 composition, i.e., each anionic SO-4 and COO(-)-binding site of CHS binds one dye cation. The composition of the saturated metachromatic complex was determined by spectrophotometric and conductometric titration of CHS with azure B, while the SO-4 and COO- content of CHS was determined by conductometric titration of CHS-acid with NaOH. The binding isotherm of azure B to CHS was determined using gelpermeation chromatography. The isotherm can be described by the model of cooperative binding of ligands to linear biopolymers. We found good agreement between theoretical predictions and experimental findings in the range of 0 less than r less than 0.8 (r = the fraction of occupied binding sites). Using a Schwarz plot, we determined the binding constants of nucleation (Kn = 2.5 X 10(3) M-1) and aggregation (Kq = 1.2 X 10(5) M-1), as well as the cooperativity parameter (q = 50), T = 295 K. With increasing CD, the strong cooperativity of the dye binding favors the formation of metachromatic aggregates rather than monomers and dimers. From the temperature dependence of Kq we evaluated the standard binding enthalpy (delta Hoq = -20.0 kJ mol-1) and entropy (delta Soq = 29.7 JK-1 mol-1) of the cooperative dye binding. The binding was found to be strongly exothermic and accompanied by a thermodynamically favorable entropy increase, this being typical of hydrophobic interactions. Solid azure B-CHS complexes were prepared according to a special dialytic technique and were studied using a microspectrophotometer equipped with a polarizer and an analyzer. The metachromatic 1:1 complex has a broad, intense absorption band whose main peak occurs at 560 nm. This corresponds with the maximum of the metachromatic dye complex in aqueous solution, i.e. 555 nm. The CHS chains of the azure B-CHS complex can be mechanically aligned in a preferred direction (k). We were able to prepare excellently orientated and very fine dye-CHS films which were birefringent and dichroic - the more birefringent, the better the mechanical orientation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Fluorescence microscopy of viable mast cells stained with different concentrations of acridine orange.

Freshly harvested rat peritoneal mast cells were stained with different concentrations of acridine orange, a metachromatic fluorochrome known to form complexes with chromatin and muscopolysaccharides. Fluorescence metachromasia was observed in cytoplasmic granules in cell populations with intracelluar dye contents as low as 5 X 10(-16) mole per cell, one-half decade lower than required to produce metachromatic staining of the nucleus. Cytoplasmic granules did not stain uniformly throughout the cell; some granules exhibited red fluorescence and others green. As the amount of acridine orange uptake per cell was increased, cytoplasmic fluorescence became uniformly red and nuclear fluorescence gradually changed from green to yellow.     

Metachromatic Agar-Diffusion Microslide Technique for Detecting Staphylococcal Nuclease in Foods

The metachromatic agar-diffusion (MAD) microslide technique was shown to detect nanogram quantities of staphylococcal thermonuclease in various foods without prior extraction, purification, or concentration.