A new method for immobilization of microbial cells by cross-linking

A method for immobilization of microbial cells was designed. The method uses generation of reactive aldehyde groups on the cell wall surface under conditions of periodate oxidation. The linking of aldehyde groups by various bifuctional aromatic diamines and then by glutaraldehyde produced immobilized cells, which are promising for use in biocatalysis with high-molecular-weight substrates.     

Periodic acid-Schiff-alcian blue: a method for the differential staining of glycoproteins

The staining mechanism underlying the periodic acid-Schiff (PAS)-Alcian Blue (AB) sequence has been investigated using a variety of glycoprotein-containing tissues from different organs of the monkey, rat and mouse. The results obtained suggest that reactive carbohydrates contain at least three types of chemical end-groups found in neutral and acidic glycoproteins: (1) PA-engendered aldehyde groups coloured magenta by the Schiff reagent; (2) PA-engendered aldehyde groups coloured blue bisulphite-AB; and (3) naturally occurring acidic (carboxyl and/or sulphate) groups coloured blue by AB only. The PAS-AB sequence showed heterogeneity of glycoprotein structures in the conjunctiva and the duodenal goblet cells. Thus, the PAS-AB sequence is not the simple reverse sequence of AB-PAS but has its own definite and unique staining selectivity and can hence be used as a reliable method for the histochemistry of glycoproteins at the light microscope level.     

Radioiodination of proteins by reductive alkylation

The use of the aliphatic aldehyde, para-hydroxyphenylacetaldehyde as the reactive moiety in the radioiodination of proteins by reductive alkylation is described. The para-hydroxyphenyl group is radiolabeled with 125I, reacted through its aliphatic aldehyde group with primary amino groups on proteins to form a reversible Schiff base linkage which can then be stabilized with the mild reducing agent NaCNBH3. The introduction of the methylene group between the benzene ring and the aldehyde group increases its reactivity with protein amino groups permitting efficient labeling at low aldehyde concentrations. Using this method, radioiodinated proteins with high specific activity can be produced. The reductive alkylation procedure is advantageous in that the labeling conditions are mild, the reaction is specific for lysyl residues, and the modification of the epsilon-ammonium group of lysine results in ionizable secondary amino groups avoiding major changes in protein charge.     

The antimutagenic activity of compounds of modified polyglucin]

The effect of modified polygluquin compounds on gamma-irradiated nuclei of V-79 Chinese hamster cells has been studied. Antimutagenic properties of the compounds are determined by aldehyde groups and oxidized chains.     

Quantification of peptide aldehyde ligands immobilized for the affinity chromatography of endopeptidases.

A method is described for quantification of aldehyde and aldehyde semicarbazone groups attached to an insoluble matrix. Semicarbazones are converted to aldehydes, and the aldehydes are coupled with 4-phenylazoaniline. The excess reagent is washed off, and the remainder then displaced with salicylaldehyde. The quantity of the phenylazoaniline/salicylaldehyde adduct is determined spectrophotometrically, allowing the calculation of the amount of aldehyde or semicarbazone per unit volume of the matrix. Analyses by the new method show that four matrices offered commercially for this type of immobilization differ greatly in coupling capacity and stability of the conjugate under conditions of affinity chromatography.     

Bacteriocidal effects and inhibition of cell separation of cinnamic aldehyde on Bacillus cereus

AIMS: In this study, bacteriocidal effects of cinnamic aldehyde on Bacillus cereus were investigated. METHODS: The bacterial culture or cell suspension in 0.85% NaCl was treated with cinnamic aldehyde at a concentration of 0.3 ml l(-1). Viable cells were counted on a nutrient agar plate. Protein leakage from the cell was determined using a protein dye. Cell morphology was observed using a scanning electron microscope. RESULTS: Bacillus cereus cells were the most sensitive to cinnamic aldehyde among four different food-borne pathogens. When the cells were treated with 0.3 ml l(-1) of cinnamic aldehyde, the viable counts decreased about 6 log cycles after 6 h of incubation. The bacterial cells remained unlysed although they were killed by cinnamic aldehyde. Treatment of cinnamic aldehyde to the exponential phase cells resulted in no significant protein leakage but strong inhibition of cell separation. CONCLUSIONS: The present findings suggest that cinnamic aldehyde exhibits bacteriocidal effects and inhibition of cell separation on B. cereus. SIGNIFICANCE AND IMPACT OF THE STUDY: These data represent an interesting background for a possible mechanism for antibacterial effects of cinnamic aldehyde.     

Studies on the molecular arrangement of RNA in tissues with a selective topo-optical reaction of RNA.

Selective demonstration of RNA in tissues was achieved by treating tissue sections with potassium permanganate followed by bisulfite and toluidine blue at pH 1.0 (PBT reaction). It is suggested that this reaction is due to aldehyde groups which are formed by the oxidative cleavage of the pyrimidine rings of RNA which can be selectively demonstrated using bisulfite-toluidine blue as the aldehyde reagent. The specificity of the reaction was tested after RNAase treatment, after acid hydrolysis, and on pure RNA droplets. The aldehyde nature of the reacting groups was checked, after permanganate oxidation, by Schiff's leucofuchsin reagent, and by aldehyde blocking reactions. Two types of intracellular molecular arrangement of RNA molecules could be distinguished by polarization optics after application of the PBT reaction: 1) The strong birefringence, dichroism and metachromatic staining of membrane-bound RNA in ergastoplasm of pancreas, liver and plasma cells indicate a linear (planar) molecular order of RNA molecules on the surface of the membranes, and 2) the isotropic, basophilic staining of RNA not organized in membrane structures (Nissl substance, nucleoli) suggest a random distribution of their dye binding sites.     

Disruption of aldehyde reductase increases group size in dictyostelium

Developing Dictyostelium cells form structures containing approximately 20,000 cells. The size regulation mechanism involves a secreted counting factor (CF) repressing cytosolic glucose levels. Glucose or a glucose metabolite affects cell-cell adhesion and motility; these in turn affect whether a group stays together, loses cells, or even breaks up. NADPH-coupled aldehyde reductase reduces a wide variety of aldehydes to the corresponding alcohols, including converting glucose to sorbitol. The levels of this enzyme previously appeared to be regulated by CF. We find that disrupting alrA, the gene encoding aldehyde reductase, results in the loss of alrA mRNA and AlrA protein and a decrease in the ability of cell lysates to reduce both glyceraldehyde and glucose in an NADPH-coupled reaction. Counterintuitively, alrA- cells grow normally and have decreased glucose levels compared with parental cells. The alrA- cells form long unbroken streams and huge groups. Expression of AlrA in alrA- cells causes cells to form normal fruiting bodies, indicating that AlrA affects group size. alrA- cells have normal adhesion but a reduced motility, and computer simulations suggest that this could indeed result in the formation of large groups. alrA- cells secrete low levels of countin and CF50, two components of CF, and this could partially account for why alrA- cells form large groups. alrA- cells are responsive to CF and are partially responsive to recombinant countin and CF50, suggesting that disrupting alrA inhibits but does not completely block the CF signal transduction pathway. Gas chromatography/mass spectroscopy indicates that the concentrations of several metabolites are altered in alrA- cells, suggesting that the Dictyostelium aldehyde reductase affects several metabolic pathways in addition to converting glucose to sorbitol. Together, our data suggest that disrupting alrA affects CF secretion, causes many effects on cellular metabolism, and has a major effect on group size.     

Studies on the molecular arrangement of RNA in tissues with a selective topo-optical reaction of RNA

Summary Selective, demonstration of RNA in tissues was achieved by treating tissue sections with potassium permanganate followed by bisulfite and toluidine blue at pH. 1.0 (PBT reaction). It is suggested that this reaction is due to aldehyde groups which are formed by the oxidative cleavage of the pyrimidine rings of RNA which can be selectively demonstrated using bisulfite-toluidine blue as the aldehyde reagent.The specificity of the reaction was tested after RNAase treatment, after acid hydrolysis, and on pure RNA droplets. The aldehyde nature of the reacting groups was checked, after permanganate oxidation, by Schiff's leucofuchsin reagent, and by aldehyde blocking reactions.Two types of intracellular molecular arrangement of RNA molecules could be distinguished by polarization optics after application of the PBT reaction: 1) The strong birefringence, dichroism and metachromatic staining of membrane-bound RNA in ergastoplasm of pancreas, liver and plasma cells indicate a linear (planar) molecular order of RNA molecules on the surface of the membranes, and 2) the isotropic, basophilic staining of RNA not organized in membrane structures (Nissl substance, nucleoli) suggest a random distribution of their dye binding sites.     

3-Formyl-1-butyl pyrophosphate A novel mycobacterial metabolite-activating human gammadelta T cells.

Most human blood gammadelta T cells react without major histocompatibility complex restriction to small phosphorylated nonpeptide antigens (phosphoantigens) that are abundantly produced by mycobacteria and several other microbial pathogens. Although isopentenyl pyrophosphate has been identified as a mycobacterial antigen for gammadelta T cells, the structure of several other stimulating compounds with bioactivities around 1000-fold higher than isopentenyl pyrophosphate remains to be elucidated. This paper describes the structural identification of 3-formyl-1-butyl-pyrophosphate as the core of several non-prenyl mycobacterial phosphoantigens bioactive at the nM range. Recognition of this molecule by gammadelta T cells is very selective and relies on its aldehyde and pyrophosphate groups. This novel pyrophosphorylated aldehyde most probably corresponds to a metabolic intermediate of the non-mevalonate pathway of prenyl phosphate biosynthesis in eubacteria and algae. The reactivity to 3-formyl-1-butyl-pyrophosphate supports the view that human gammadelta T cells are physiologically devoted to antimicrobial surveillance.     

Separation of glutaraldehyde and some of its aldol condensation products by hydroxyl-aldehyde group affinity chromatography

Glutaraldehyde exists in aqueous solution in equilibrium with monomers and polymers of cyclic and open-chain hemialdals and hydrates. At alkaline pH oligomeric and polymeric alpha,beta-unsaturated aldehyde derivatives are formed from primarily produced aldol condensation products. This communication reports a method for separation of such aldol condensates by means of a new high performance liquid chromatography technique based on the affinity of aldehyde groups for hydroxyl groups of a hydroxylated polyether matrix (Bio-Gel SEC-10). Five peaks corresponding to different aldol condensates of glutaraldehyde were obtained from the affinity column. They have been distinguished by 1H-NMR and UV spectroscopy. Kinetic measurements yielded formation rates for the different aldol condensates.     

4-Hydroxynonenal interacts with tubulin by reacting with its functional -SH groups

4-Hydroxynonenal, which is one of the most important products of lipid peroxidation, alters microtubular organization and structure in 3T3 fibroblasts. Changes in cell shape and the disappearance of microtubules are observed by immunofluorescence after incubation with the aldehyde, and the colchicine binding activity of tubulin from 3T3 cells is modified. Moreover, the aldehyde determines a decrease in the ability of purified tubulin to polymerize and to bind colchicine. These effects may be related to the interaction of the aldehyde with functional -SH groups of tubulin which are necessary for protein integrity and functions. Indeed, the addition of cysteine protects against the damaging effects of the aldehyde.     

The oxidation and (methylthio)methylation of cotton by methyl sulfoxide-acetic anhydride

Methyl sulfoxide-acetic anhydride oxidizes some of the cellulosic hydroxyl groups of cotton yarn to aldehyde and ketone groups, while etherifying other hydroxyl groups with (methylthio)methyl groups. The ratio of oxidation to (methylthio)methylation of the cellulose is influenced by the reaction temperature, the reaction time, and the ratio of methyl sulfoxide to acetic anhydride employed. The (methylthio)methyl groups may be cleaved by boiling the cotton in aqueous solutions of acetic acid.The total carbonyl content of the oxidized cellulose was determined by conversion into the polyoxime. The ratio of aldehyde to ketone groups in the oxidized cellulose was determined by further oxidation with chlorous acid; the car?yl groups produced were measured spectrophotometrically by a method involving adsorption of Methylene Blue.     

4-hydroxy-2,3 trans-nonenal nuclear concentration inversely correlates with the growth rate of T leukemia cells

4-hydroxy-2,3 trans-nonenal is the major diffusible product generated by linoleic and arachidonic acids peroxidation. Its endogenous content is inversely related to the rate of cell proliferation and directly related to the level of cell differentiation. As previously reported, the nuclear localization of the aldehyde has been observed by means of a fluorescent antibody and confocal microscopy, and its concentration measured by electrospray/mass spectrometry, a sensitive and selective method for 4-hydroxy-2,3 trans-nonenal determination, on nuclear extracts of leukemic cells. With the aim to establish a possible correlation between the peroxidation product nuclear concentration and cell growth rate, Jurkat 6 leukemic cells have been used and the aldehyde measured by electrospray/mass spectrometry. The cells arrested in G1 show a content of 4-hydroxy-2,3 trans-nonenal significantly increased with respect to control ones.     

Staining properties of aldehyde fuchsin analogs.

This investigation was designed to clarify the role of the aldehyde component of aldehyde fuchsin in its staining reactions. Several aldehyde fuchsin analogs were prepared by using different aldehydes. The staining quality of these analogs and pararosaniline-HCl was compared with that of aldehyde fuchsin prepared with paraldehyde in the usual way. The major findings of this investigation include: 1) Aldehyde fuchsin staining of nonoxidized pancreatic B cells requires a stain prepared with either paraldehyde or acetaldehyde. 2) An aldehyde moiety is required for aldehyde fuchsin staining of strong tissue anions. 3) Staining of elastic tissue with aldehyde fuchsin analogs resembles staining of strong tissue anions more than staining of nonoxidized pancreatic B cells. Possible reaction mechanisms of aldehyde fuchsin with tissue substrates are discussed.     

Concurrent Demonstration of Desoxyribose Nucleic Acid and 1,2-Glycols

Sections from rat tissue were fixed in 10% formalin in 90% alcohol and placed in a 1.0% suspension of sodium bismuthate (NaBiO₃) in 40% phosphoric acid for 40 minutes at room temperature. Bismuth phosphate crystals were removed with 2N HCl. The sections were next placed in the Schiff reagent for 20 minutes. By this method the DNA was hydrolyzed by the phosphoric acid and the 1,2-glycols were oxidized by the NaBiO₃. In both cases aldehyde groups were released and subsequently stained by the Schiff reagent. A photomicrograph is included demonstrating the nuclei, goblet cells, striated border and basement membrane stained by this combined method.     

Hydride transfer stereospecificity of rat liver aldehyde dehydrogenases

The stereospecificity of hydride transfer to NAD+ by several forms of rat liver aldehyde dehydrogenase was determined by a nuclear magnetic resonance method. The forms included several mitochondrial and microsomal isozymes from normal liver, as well as isozymes from xenobiotic-treated and tumor cells. The proton added to NAD+ comes exclusively from the aldehyde substrate and in all cases was A (pro-R)-stereospecific.     

Influence of some aldehyde blocking agents on staining of depurinized DNA with cationic dyes.

Rat liver, spleen and Walker carcinosarcoma imprints were subjected to depurinizing Feulgen hydrolysis and then treated with blocking agents of aldehyde groups. Such blockators as sodium bisulfite and hydroxylamine which multiplay additionally anionic groups in DNA and intensify the reactions with cationic dyes, ensuring anisotropic staining. Hydrazine lowers the binding of carionic dyes to DNA, instead phenylhydrazine, completely blocks both aldehyde and phosphate groups. When the imprints were treated with 2.4-dinitrophenylhydrazine, aldehyde and phosphate groups of apurinic acid were blocked, and DNA staining by cationic dyes occurred only on account of nitrogroups of the blocking agents which have been used. The staining reaction of cationic dyes after the use of anionogenic blocking agents of aldehyde groups is prospective not only for revealing DNA but also for several other compounds with natural or potential aldo- and ketogroups. However the reaction with phenylhydrazine can serve as a staining without removal of DNA prior to staining as an optional procedure.     

Quantitative aspects of the Ninhydrin-Schiff reaction on amino cellulose films and tissue sections

Summary In the ninhydrin-Schiff reaction primary amino groups are converted by oxidation with ninhydrin to aldehyde groups which are subsequently stained with pararosanilin. Amino cellulose films, used as a model, and sections of muscle tissue were submitted to this reaction. The amino groups were stained before and after the ninhydrin reaction with dinitrofluorobenzene and the generated aldehyde groups were stained with dinitrophenyl-hydrazine. The molar extinction coefficients used for the calculation of the molar amounts of chromophores from extinction values, and the conditions for maximal staining intensity were determined on the amino cellulose model. With these data the yields of the different steps in the reaction sequence could be calculated in molar amounts from the extinction measurements. The results showed that from the amino groups originally present in the tissue sections less than 40% were converted by ninhydrin. About 90% of the converted amino groups were found as aldehyde groups and from these only 7% reacted with pararosanilin. On amino cellulose similar data were obtained. Attempts were made by modification of the conditions in the ninhydrin oxidation step to increase the overall yield of the reaction. These were only partially successful, but indicate that further quantitative study of other reaction conditions and different aldehyde generating agents could be promising.     

New substrates of polyamine oxidase. Dealkylation of N-alkyl-alpha, omega-diamines

1) N-Alkyl-alpha, omega-diaminoalkanes are substrates of polyamine oxidase. 2) The compounds are oxidatively cleaved and form equimolar amounts of an aldehyde, a diamine, and H2O2. 3) Minimum structural requirements of a substrate of polyamine oxidase are two positively charged amino groups and an alkyl-substituent on one or both nitrogen atoms. 4) Dealkylation of N-alkyl derivatives by polyamine oxidase in vivo is a method to accumulate diamines in brain, and to release intracellularly an aldehyde from a stable prodrug.