Polyacrylamide films as a tool for investigating qualitative and quantitative aspects of the staining of glycosaminoglycans with basic dyes

Synopsis With the introduction of model films of polyacrylamide gel into which purified glycosaminoglycans (GAGs) have been incorporated, the direct recording of metachromatic spectra with virtually no interference of the corresponding orthochromatic peaks has become possible. Because this model system yields situations comparable to those of stained sections under the microscope, it is well suited for investigating qualitative and quantitative aspects of histochemical staining procedures. Previous model experiments have shown that under aqueous conditions only minor differences can be observed between the metachromatic peaks of different GAGs complexed with a suitable dye (e.g. Toluidine Blue O, Thionin, Safranin O, Cresyl Violet, Crystal Violet). In non-aqueous media, such as glycerol and ethylene glycol, the complexes with Toluidine Blue O revealed a special pattern for heparin, having a metachromatic peak (517 nm) about 30 nm lower than that of all other GAGs. This observation has formed the basis of a method for the qualitative microspectro-photometric detection of heparinin situ which was worked out by combining model film experiments with microspectrophotometric data obtained from rat mast cells. Since only a limited number of cells is necessary for obtaining reliable data with this method, the presence of heparin in the cytoplasmic granules of normal human mast cells and basophilic granulocytes could thus be proved directly.Alcian Blue 8GX, another basic dye frequently used in GAG histochemistry, has also been investigated with polyacrylamide films. In contrast to the metachromatic dyes, the rate of staining with Alcian Blue depends to a large extent on the rate of penetration of the dye into the model films. The rate of penetration is also a phenomenon of great importance for dye bindingin situ, where complex basic protein molecules may form a barrier for the Alcian Blue molecules. The model film studies performed so far have yielded conditions that provide maximal staining (up to an optimal level) and a linear relationship between the concentration of GAG and the AB binding. The presence of basic protein, electrostatically bound to the GAG, was not found to influence either the rate of staining or the maximal amount of dye binding.Paper presented at a symposium The Changing directions of carbohydrate histochemistry, at the fifth International Congress of Cytochemistry and Histochemistry in Bucharest, Romania on 1 September 1976.     

Genetic and functional analyses of the primary in vitro CTL: Response of NZB lymphocytes toH-2-compatible cells

Spleen cells from NZB mice make an unexpected primary cytotoxic T lymphocyte (CTL) response to BALB/c cells in vitro. In this study, it is shown that this response is comprised of at least three independent components. These include a response to antigens recognized in association with H-2^d products, a response to Qa-1^b-associated antigens which is notH-2-restricted and a response directed toward antigens not associated with either H-2^d- or Qa-1^b-coded determinants. The last response appears to be the weakest of the three. In addition, cells from NZB F₁ mice which were either homozygous (Qa-1 ^a /Qa-1 ^a ) or heterozygous (Qa-1 ^a /Qa-1 ^b ) forQa-1 alleles, all responded to BALB/c cells. These data suggest that the NZB CTL response to BALB/c cells is not solely dependent on antigens coded for by genes in theH-2D-Tla region for either the sensitization or effector phases of the response. The ontogeny of the NZB anti-BALB/c CTL response coincides with that of a number of B-cell abnormalities but is shown in experiments with-suppressed NZB mice to be independent of B-cell dysfunction. Studies with (NZB x B10.D2)F₁ + B10.D2 mice demonstrated that the anti-BALB/cCTL response to antigens coded for outside ofQa-1 is governed by at least two genes. Finally, it is shown that another conventionallyH-2-restricted response, that to TNP-modified isologous cells, is neither significantly cross-reactive nor markedly elevated in NZB mice. — The foregoing observations suggest that some subsets of NZB T lymphocytes are intrinsically abnormal. The possibilities that the apparent hyperreactivity of NZB CTL precursors, evidenced in the response to BALB/c cells, is primary or results from the secondary effects of excess T-cell help are discussed.     

Calmodulin binding to platelet plasma membranes

Calmodulin copurifies with platelet plasma membranes isolated by glycerol-induced lysis and density gradient centrifugation. These membranes also bind ¹²⁵I-labeled calmodulin in vitro in the presence of Ca²⁺. Binding is largely reduced by replacing Ca²⁺ by Mg²⁺ or by addition of an excess unlabeled calmodulin. The specific component of binding is saturable, with an apparent K_d of 27 nM and a maximum of 15.9 pmol binding sites per mg of membrane protein. This is equivalent to approx. 4100 binding sites per platelet. Binding was inhibited by addition of phenothiazines, a group of calmodulin antagonists. Half-maximal inhibition was attained with approx. 20 μM trifluoperazine or 50 μM chlorpromazine. In contrast, chlorpromazine-sulfoxide which is inactive towards calmodulin, did not affect the binding. Calmodulin binding polypeptides of the plasma membrane were identified by a gel-overlay technique. A major calmodulin-binding component of molecular weight 149 000 was detected. Binding to this band was Ca²⁺-dependent and inhibited by chlorpromazine. The molecular weight of this polypeptide is similar to that of glycoprotein I and also that of the red cell (Ca²⁺ + Mg²⁺)-stimulated ATPase, which is known to bind calmodulin. The possible role of calmodulin in platelet activation is analysed.     

The identification of dimethylphenylarsine as a microbial metabolite using a simple method of chemofocusing

Candida humicola acts on benzenearsonic acid to produce dimethylphenylarsine, which was identified by mass spectroscopy following the chemofocusing of the volatile metabolite onto a mercuric chloride impregnated filter. The same technique established that trimethylarsine is the volatile metabolic product obtained from C. humicola treated with 4-NH₂-2-OHC₆H₃AsO(OH)₂ and (CH₃)₃AsO. Arsanilic acid, 4-NH₂C₆H₄AsO(OH)₂, is not metabolized to a volatile arsine.     

Modification of γ-Aminobutyric AcidA Receptor Binding and Function by N-Ethoxycarbonyl-2-Ethoxy-1,2-Dihydroquinoline In Vitro and In Vivo: Effects of Aging

The irreversible protein-modifying reagent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) was used to investigate binding site characteristics on the gamma-aminobutyric acidA (GABAA) receptor complex. In vitro, preincubation with EEDQ led to a concentration-dependent decrease in receptor number for benzodiazepine, t-butylbicyclophosphorothionate (TBPS), and GABA binding sites in cerebral cortex. The effect was maximal at the highest concentration of EEDQ used (10(-4) M) and was greatest for the benzodiazepine site. Pretreatment of membranes with the benzodiazepine antagonist Ro 15-1788, 1 or 10 microM, or the agonist lorazepam, 10 microM, largely prevented the effects of EEDQ. Scatchard analysis indicated no effect of EEDQ, 10(-4) M, on apparent affinity, but a decrease in receptor density for each site. Administration of EEDQ to mice, 12.5 mg/kg i.p., led to a substantial (55-65%) decrease in number of benzodiazepine binding sites in cortex after 4 h. Slightly smaller changes were observed for TBPS and GABA binding. No changes were observed in apparent affinity at any site. Prior administration of Ro 15-1788, 5 mg/kg, prevented the effect of EEDQ on benzodiazepine binding. Density of benzodiazepine binding sites gradually recovered over time, and receptor density returned to control values by 96 h after EEDQ injection. Number of binding sites in cortex for TBPS and GABA also increased over time after EEDQ. Benzodiazepine sites in cerebellum were decreased proportionally to cortex after EEDQ, and increased over a similar time course. Function of the GABAA receptor in chloride uptake in cortex was markedly reduced (65%) by EEDQ.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alterations in Cortical [3H]Kainate and α-[3H]Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid Binding in a Spontaneous Canine Model of Chronic Hepatic Encephalopathy

Excitatory amino acid receptor binding parameters were investigated in a spontaneous dog model of chronic hepatic encephalopathy. L-[3H]Glutamate, (+)-[3H]-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-im ine maleate ([3H]MK-801), [3H]kainate, and alpha-[3H]-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ([3H]AMPA) binding experiments were performed using crude cerebrocortical synaptosomal membrane preparations from dogs with congenital portosystemic encephalopathy (PSE) and control dogs. There was no change in the affinity or density of L-[3H]-glutamate or [3H]MK-801 binding sites in dogs with congenital PSE compared with control dogs. However, in the PSE dogs there was a significant reduction in the density of [3H]kainate binding sites compared with control dogs and abolition of the low-affinity [3H]AMPA binding site. The relative binding capacity of PSE synaptosomal membranes for [3H]kainate and [3H]AMPA was expressed as the ratio Bmax/KD. There was a significant inverse correlation between the Bmax/KD ratio for [3H]AMPA binding and the worst grade of encephalopathy experienced by each dog. These results suggest that there is a significant perturbation of cerebrocortical non-N-methyl-D-aspartate receptor binding in dogs with congenital PSE which may have relevance to the pathogenesis of hepatic encephalopathy.     

Glutamine Synthetase in Oligodendrocytes and Astrocytes: New Biochemical and Immunocytochemical Evidence

The results of recent immunocytochemical experiments suggest that glutamine synthetase (GS) in the rat CNS may not be confined to astrocytes. In the present study, GS activity was assayed in oligodendrocytes isolated from bovine brain and in oligodendrocytes, astrocytes, and neurons isolated from rat forebrain, and the results were compared with new immunochemical data. Among the cells isolated from rat brain, astrocytes had the highest specific activities of GS, followed by oligodendrocytes. Oligodendrocytes isolated from white matter of bovine brain had GS specific activities almost fivefold higher than those in white matter homogenates. Immunocytochemical staining also showed the presence of GS in both oligodendrocytes and astrocytes in bovine forebrain, in three white-matter regions of rat brain, and in Vibratome sections as well as paraffin sections.     

Aboriginal preparation ofCycas seeds in Australia1

The seeds of cycad plants are a toxic food used by many Aboriginal groups in northern Australia. Acute symptoms produced after consumption of untreated Cycas seeds are due to azoxyglycosides, especially cycasin, although the toxic dose depends on the animal species tested. There are three traditional methods used to treat these seeds: brief leaching in water; prolonged leaching in water; and aging. Aboriginal people living at Donydji outstation in northeast Arnhem Land, most regularly consume aged seeds ofCycas angulata R.Br. Analyses of fresh seeds and seeds prepared at Donydji and in the laboratory indicate that cycasin is effectively removed by all the traditional preparation techniques, although each technique has an end product with different storage and handling properties. The social implications of processing need further elaboration, but these techniques have a long history and archaeological remains of seeds in Australia may date back to the Pleistocene.     

Synthesis of 2-chloro-2′-deoxyadenosine by washed cells ofE. coli

Summary Washed cells ofE. coli ATCC 5275, a thymine auxotroph, catalysed formation of 2-chloro-2-deoxyadenosine when incubated with 2-chloroadenosine and a variety of deoxynucleosides. This transdeoxyribosylation reaction was complete after 4 h of shaking at 37°C. The equilibrium reaction mixture favoured product formation when purine rather than pyrimidine deoxyribonucleosides were used as cosubstrates, and when the ratio of deoxysugar donor to 2-chloroadenosine was high. Using deoxyadenosine as cosubstrate, chlorodeoxyadenosine was purified from larger scale reaction mixtures by treatment with Dowex-1 (OH-form) or by high performance liquid chromatography.