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Determination of proteins using the p-acetylchlorophosphonazo-barium(II) complex as a spectroprobe.

A novel spectrophotometric method has been developed for the determination of proteins using the p-acetychlorophosphonazo (CPA-pA)-barium(II) complex as a spectroprobe. In the pH range of 2.0-2.8, the absorbance of the CPA-pA-barium(II) complex at 649 nm is greatly decreased by protein. The absorbance decrease is in proportion to the concentration of protein in the range of 0-20 mg/L. The apparent molar absorptivities are 1.56 x 10(6), 1.70 x 10(6) and 6.04 x 10(5) L mol(-1) cm(-1) for bovine serum albumin (BSA), human serum albumin (HAS) and ovalbumin (OVA), respectively. The method is reproducible and simple, and has been used to determine total protein in human sera. The results are in agreement with those obtained by the pyrocatechol violet (PV)-Mo(VI) method, with relative standard deviations of 2.5-3.5% (n=6).

Barium↗

Sensitive spectrophotometric determination of phosphate using silica-gel collectors.

Phosphate, 3 - 10 nmol, in 1 dm3 of natural-water samples was quantitatively collected along with 10 micromol of Fe(III) ion onto a silica-gel collector in the pH range of 5.4 - 6.2. The amount of Fe(III) ion needed was limited to such a low level that the Pyrocatechol Violet method could be applied without removing the Fe(III) ion, providing a superior determination method for phosphate. Surface-water samples of the north basin of Lake Biwa and the Tanabe Bay in the Wakayama prefecture were selected as being representative of natural water with extremely low phosphate concentrations, and were found to have phosphate concentrations of 3.68 and 4.31 nmol dm(-3), respectively.

Journal Article↗

Catechol and noradrenaline facilitate the neuromuscular transmission via different mechanisms.

Using curarized frog sartorius muscles, selective presynaptic facilitatory effects of two structurally related chemicals, noradrenaline (NA) and catechol (pyrocatechol, PC), were compared by calculating the fractional release (P) from the rundown of endplate potential during a train-of-three stimulation. Below 10 microM, both NA and PC increased P in a similar manner. At higher concentrations, however, the effects of NA were found to be quite different from those of PC. NA never increased further the value of P (0.11-0.13) whereas PC augmented P in a dose dependent fashion up to about 0.76. Thus, the present data suggested that NA and PC facilitate the transmitter release via different mechanisms. The effects of the latter were identical to the case of "chemical potentiation or accelerated evoked release" by 4-aminopyridine.

Animals↗

2(3H)-benzoxazolone and bioisosters as "privileged scaffold" in the design of pharmacological probes.

The 2(3H)-benzoxazolone heterocycle and its bioisosteric surrogates (such as 2(3H)-benzothiazolinone, benzoxazinone, etc.) have received considerable attention from the medicinal chemists owing to their capacity to mimic a phenol or a catechol moiety in a metabolically stable template. These heterocycles and pyrocatechol have indeed similar pKa's, electronic charge distribution, and chemical reactivity. Therapeutic applications of this template are very broad, and range from analgesic anti-inflammatory compounds (including PPAR-gamma antagonists) to antipsychotic and neuroprotective anticonvulsant compounds. High affinity ligands have been obtained also for dopaminergic (D2 and D4), serotoninergic (5-HT1A and 5-HT-2A), sigma-1 and sigma-2 receptors. Owing to the high number of positive hits encountered with this heterocycle and its congeners, 2(3H)-benzoxazolone template certainly deserves the title of "privileged scaffold" in medicinal chemistry.

Benzoxazoles↗

Transneuronal transport of wheatgerm agglutinin conjugated with horseradish peroxidase in the somatosensory system of the rat: a light- and electron-microscopic study.

Experiments were performed in order to investigate, at the light- and electron-microscopic levels, the transneuronal transport of wheatgerm agglutinin conjugated with horseradish peroxidase (WGA:HRP) in the somatosensory system of rats. In five adult albino rats, various amounts of WGA:HRP at different concentrations were bilaterally injected in the dorsal column nuclei (DCN). In one additional animal, WGA:HRP was injected in one side, whereas free HRP was injected in the contralateral DCN. In another five rats, WGA:HRP was injected in the first somatosensory cortex (SI). The postinjection survival time of the animals ranged from 30 to 48 hr. The histochemical visualization of the enzyme was performed using either paraphenylenediamine-pyrocatechol (PPD-PC) or tetraethylbenzidine (TMB) as chromogens on adjacent horizontal serial sections. All the reacted samples were studied at the light-microscopic level, and sections from four animals were processed for the ultrastructural investigation. After DCN injections, a massive anterograde labeling was always observed in nucleus ventralis posterolateralis (VPL) of the thalamus, where also labeled neurons and glial cells were detected at both the light- and the electron-microscopic levels. Labeled neurons and terminals in the lateral border of nucleus reticularis (Re) of the thalamus were also observed after either DCN or SI injection of WGA:HRP. Our results show that WGA:HRP is effectively transported not only anterogradely and retrogradely through the somatosensory system of the rat, but also transneuronally. The transneuronal transfer of the tracer seems to be mainly related to the postlabeling survival time of the animal, and it does not occur when free HRP is injected. In controlled experimental conditions, WGA:HRP can therefore be useful for tracing secondary projections. Moreover, in the present report, the existence of a mediolateral arrangement of the projections of the somatosensory-related area of Re toward VPL is directly demonstrated. As for the histochemical procedure employed, differences in sensitivity are shown between PPD-PC and TMB, although the same general pattern of labeling is present with both chromogens.

Afferent Pathways↗

The intrinsic organization of the ventroposterolateral nucleus and related reticular thalamic nucleus of the rat: a double-labeling ultrastructural investigation with gamma-aminobutyric acid immunogold staining and lectin-conjugated horseradish peroxidase.

An electron-microscopic investigation of the synaptic organization of the rat's ventroposterolateral nucleus (VPL) and of a reticular thalamic nucleus (RTN) area related to somatosensory thalamic nucleus was performed. In a group of 11 rats, wheatgerm agglutinin conjugated to horseradish peroxidase (WGA:HRP) was injected either in the first somatosensory area of cortex (SI) or in the dorsal column nuclei (DCN). The retrogradely and/or anterogradely transported enzyme was visualized using paraphenylenediamine-pyrocatechol (PPD-PC) as substrate. In a second series of six experiments, an immunocytochemical procedure using a specific anti-gamma-aminobutyric acid (anti-GABA) was employed. Postembedding localization of GABA was performed for ultrastructural observation by means of the colloidal gold immunostaining procedure. Thin sections of recognized VPL and RTN areas from WGA:HRP-injected animals were further processed for immunocytochemistry in order to localize simultaneously, at the electron-microscopic level, the transported enzyme and GABA. The results obtained with this procedure demonstrated that HRP-labeled terminals from DCN contacted the soma and proximal dendrites of VPL neurons, while the terminals labeled after SI cortical injections were predominantly localized to the distal portion of the dendrites. The same cortical injection also determined the presence of labeled synaptic boutons contacting the soma, and both proximal and distal dendrites of RTN neurons. GABA-immunolabeled terminals were observed in VPL in a number larger than those observed with other methods, since not only typical F terminals were labeled but also terminals containing round and/or pleomorphic vesicles. GABA-ergic terminals contacted the soma and the proximal and distal dendrites of VPL neurons, while in RTN cells they made synaptic contact mainly with the soma and proximal dendrites. In the double-labeling experiments, terminals containing both HRP and specific immunogold GABA staining were never observed. The present data provide a direct demonstration of the presence of a strong inhibitory input from RTN upon VPL neurons and of the existence of autoinhibition within RTN neurons.

Animals↗

Comparison between two chromogenic substrates for revealing an immunoperoxidase reaction of human metaphase chromosomes.

To study the binding of an antiadenosine serum to human chromosome DNA, two types of chromogenic reagents were compared. The procedure is as follows: lymphocytes with metaphase chromosomes were spread on slides; some slides were irradiated by UV; all slides were then incubated with antiadenosine rabbit serum and then with antirabbit sheep serum labelled with peroxidase; the latter was revealed in the classical manner by 3,3'-diaminobenzidine (DAB), or, alternatively, by p-phenylenediamine plus pyrocatechol (PPD-PC). The present study shows that the results obtained with PPD-PC were equivalent, if not superior, to those obtained with DAB. In the case of weaker reactions, results with PPD-PC were superior. Furthermore, this reagent has the major advantage of being noncarcinogenic.

3,3'-Diaminobenzidine↗

Appraisals of compounds of diverse chemical classes for capacities to cure infections with sporozoites of Plasmodium cynomolgi.

Compounds (265) of widely diverse structures were appraised for radical curative activity in rhesus monkeys infected with sporozoites of the B strain of Plasmodium cynomolgi, using an evaluation system that provided a preliminary assessment with from 0.1-1.0 g of compound and tests against one to five active infections. None of 32 compounds in a miscellaneous structure category, none of seven agents of antibiotic origin, none of 12 1,5-naphthyridines, and none of seven 7-aminoquinolines exhibited curative activity at the largest test doses. There was a suggestion that one of 12 newly synthesized pyrocatechols was curative. Two of 20 6-aminoquinolines effected cure at or near maximum tolerated doses. In contrast, 90 of 174 newly synthesized 8-aminoquinolines effected cure; 18 of the 90 being as active as primaquine, eight twice as active, and six four times as active. There were major disagreements between the above results and those recorded by others in mice inoculated with sporozoites of P. berghei yoelii or P. yoelii nigeriensis. These discrepancies were of serious dimensions in evaluations of the 8-aminoquinolines. This, plus previous near flawless performances of P. cynomolgi in identifying agents that would cure naturally acquired P. vivax infections, led to the suggestion that the abbreviated simian model employed in these studies be used hereafter in primary screening of new agents for radical curative activity.

Aminoquinolines↗

[Spectrophotometric and fluorometric study of albumins and hemin interactions with aromatic antioxidants].

Difference spectrophotometry and fluorescence quenching of human and bovine serum albumins were used to determine their association constants (Ka) with hemin in buffered physiological saline (pH 7.4) supplemented with 2% dimethylsulfoxide or in 40% aqueous dimethylformamide (pH 7.4). Ka values depended on the medium, the extent of albumin delipidation, and on the method of determination. The formation of hemin complexes with o-phenylenediamine, tetramethylbenzidine, gallic acid, its polydisulfide, and two substituted di-tert-butyl pyrocatechols was studied by difference spectrophotometry in the same media; Ka values for the complexes were calculated and compared to each other. The formation of complexes of these aromatic ligands with albumins was studied fluorometrically; Ka values were of order of approximately 10(-5) M-1 and decreased with the ligand hydrophobicity.

Animals↗

[Certain biochemical and physicochemical properties of the inducible form of extracellular laccase from basidiomycetes Coriolus hirsutus].

An inducible form of extracellular laccase (EC 1.14.18.1) was isolated from the basidiomycete Coriolus hirsutus. The induction was performed with 0.11 microM syringaldazine, a substrate of laccase. The inducible form of the enzyme consisted of two isoforms, laccase I1 and laccase I2, whose molecular weights were 69 +/- 2 and 67 +/- 2 kDa, respectively. The isoelectric points of these isoenzymes were found to be 3.5 and 4.2, respectively. The optimum pH range for both laccases was 4.4-4.6, and the optimum temperature was 50 degrees C. The thermal stability of these isoenzymes was examined, and KM values for the substrates syringaldazine and pyrocatechol were determined. Our biochemical and physicochemical studies demonstrated that inducible laccase isoforms differed from constitutive forms in molecular weight, IP, KM, and thermal stability. However, their optimum pH ranges and temperatures were identical.

Electrophoresis, Polyacrylamide Gel↗

Mn-peroxidase from Bjerkandera adusta 90-41. Purification and substrate specificity.

Mn-peroxidase has been purified to homogeneity from culture liquid of white-rot fungus Bjerkandera adusta 90-41 grown on medium containing lignosulfonates. According to the data on SDS-PAGE and isoelectrofocusing, the molecular mass of the enzyme is 43 kD and the isoelectric point is 3.5. The pH-optimum in the reaction of MnSO4 oxidation is 4.5. The substrate specificity of the enzyme has been studied. In contrast to previously known Mn-peroxidases from B. adusta, the isolated enzyme has no activity with veratryl alcohol. The enzyme can oxidize ammonium 2, 2-azino-bis(ethyl-6-benzothiazoline sulfonate) (ABTS), o-phenylenediamine, and phenol red in the absence of Mn2+. Oxidation of ABTS and o-phenylenediamine is stimulated by Mn2+, whereas in the reaction of oxidation of phenol red Mn2+ acts as an inhibitor. Some aromatic substrates, such as pyrocatechol and guaiacol, are oxidized only in the presence of Mn2+.

Chromatography, Ion Exchange↗

The chemotherapy of rodent malaria, XXIII Causal prophylaxis, part II: Practical experience with Plasmodium yoelii nigeriensis in drug screening.

Data are presented on the causal prophylactic action of about 100 compounds of various types against Plasmodium yoelii nigeriensis N67 in mice. Examples are given to show how action against pre-erythrocytic schizonts may be differentiated from action on emerging erythrocytic stages. In a series of 35 8-aminoquinolines, all but 10 showed definite causal prophylactic activity at tolerated doses. The data permit the compounds to be ranked in order of activity, and many are shown to be more active in this test system than primaquine. Marked causal prophylactic activity is displayed by a variety of quinone structures, several of which show a significant residual action on blood stages. A high level of activity is found in dihydrofolate reductase inhibitors within several chemical classes. Rorguanil is more effective as a causal prophylactic than a blood schizontocide in the mouse as in man. Sulphonamides and sulphones are also effective in this system. The active levels are influenced by the content of PABA in the diet of the hosts. Causal prophylactic action has been detected in a number of experimental compounds including some antibiotics (such as tetracycline and clindamycin). The pyrocatechol RC 12 shows only slight activity at the maximum tolerated dose. Chloroquine, mepacrine, quinine, quinolinemethanols and phenanthrenemethanols are inactive as causal prophylactics. It is concluded that a rodent malaria-mouse model does provide a relatively simple model for the screening of drugs for causal prophylaxis, and the data so obtained are of relevance to the detection of causal prophylactics against human malaria.

Amidines↗

[Melanin complex of the fungus Inonotus obliquus].

The fungus Inonotus obliquus (Pers.) Pil. synthesised high-molecular-weight phenolic pigments that were assigned to melanins according to their physicochemical properties. It was showed that copper ions (0.008%), pyrocatechol (1.0 mM), and tyrosine (20.0 mM) stimulated the melanogenesis. The production of melanin correlated with the synthesis of o- and p-diphenoloxidases. The fungal melanin had strong antioxidant and genoprotective effects.

Antimutagenic Agents↗

[Growth of Rhodococcus opacus on mixtures of monohalogenated benzenes and phenols].

The growth of Rhodococcus opacus GM-14 on mixtures of 2-chloro- and 2-bromophenol, of 4-chloro, 4-bromo-, and 4-iodophenol, and of chloro-, bromo-, and iodobenzenes was accompanied by consumption of the substrates and excretion of halogen ions to the medium. During the growth on monochlorophenols, the substrates were consumed sequentially in the following order: 4-chloro-, 3-chloro-, and then 2-chlorophenol. Chlorine ions were excreted in a two-phase manner in amounts comprising 79% of the theoretical yield. The diauxic growth of R. opacus GM-14 can be explained by the existence in this bacterium of two modified metabolic pathways for the ortho-cleavage of halogenated pyrocatechols. The first pathway included 4-halogeno- or dihalogenopyrocatechols as intermediates, whereas the second pathway included 3-halogenopyrocatechols.

Benzene Derivatives↗

A new oxygenase, 2-nitropropane dioxygenase of Hansenula mrakii. Enzymologic and spectrophotometric properties.

2-Nitropropane dioxygenase, purified to homogeneity from Hansenula mrakii (IFO 0895), has a molecular weight of approximately 62,000 and consists of two subunits nonidentical in molecular weight (39,000 and 25,000). Stoichiometrical studies and the results obtained with 18O2 showed that 2 atoms of molecular oxygen are incorporated into 2 molecules of acetone formed from 2-nitropropane. In addition to 2-nitropropane, nitroethane, 3-nitro-2-pentanol, and 1-nitropropane are oxidatively dentrified. The enzyme, which exhibits absorption maxima at 274, 370, 415, and 440 nm and a shoulder at 470 nm, contains 1 mol of FAD and 1 g atom of non-heme iron per mol of enzyme. The enzyme-bound FAD is reduced by 2-nitropropane under anaerogic conditions, but the enzyme-bound Fe3+ is not affected. The introduction of oxygen to the reduced form of enzyme causes reoxidation of the enzyme. The bound FAD and Fe3+ are reduced by the addition of nitromethane, which is not a substrate, under anaerobic conditions. The aerobic dialysis of the enzyme treated with nitromethane causes reoxidation of only the Fe2+. Sodium dithionite also reduces both the enzyme-bound FAD and Fe3+ under anaerobic conditions. When the enzyme is dialyzed against 10 mM potassium phosphate buffer (pH 7.0) immediately after reduction by dithionite, the absorption spectrum similar to that of the native enzyme appeared with concomitant restoration of approximately 80% of the activity. The enzyme activity is significantly inhibited by pyrocatechol-3,5-disulfonate disodium salt, 8-hydroxyquinoline, reducing agents such as 2-mercaptoethanol, and HgCl2. The Michaelis constants are as follows: 2-nitropropane (2.13 X 10(-2) M), nitroethane (2.43 X 10(-2) M), 3-nitro-2-pentanol (6.8 X 10(-3) M), 1-nitropropane (2.56 X 10(-2) M), and oxygen (3.03 X 10(-4) M, with 2-nitropropane).

Amino Acids↗

[Effect of cyclic compounds on pigment formation in Aspergillus niger cultures].

The mycelial pigment os Asp. niger VP has been fractionated into four fractions ehose solubility is similar to melanin pigments obtained from other sources. The addition of pyrocatechol, L-aspartic, benzoic, kojic and salicylic acids to the liquid Capek medium stimulates the fungal growth but diminishes the yield of fractions of the melanin pigment from the mycelium. Cinnamic acid does not influence the growth but also decreases the yield of the pigment. DL-tyrosine stimulates the fungal growth and produces no influence on the total yield of fractions 2, 3 and 4 from the mycelial pigment. All the cyclic compounds enhance the pigmentation of the culture liquid and give rise to the formation of fractions 2 and 3 of the melanin pigment. This latter constitutes a substantial portion of the dry matter of the fungal cell. The pigment content in the mycelium is much higher than that in the culture liquid of the corresponding culture.

Aspartic Acid↗

[Sensitization to resorcinol in a prescription verrucide preparation: unusual systemic clinical features and prevalence].

BACKGROUND: Resorcinol is a rare sensitizer. In Lorraine, where it is used in high concentrations in an anti-wart ointment, this molecule has induced many cases of allergy. The purpose of this study was to describe clinical features in patients sensitized to resorcinol, to determine the incidence of sensitization in Lorraine and ascertain the occurrence of cross reactions with other phenolic pro-haptens. PATIENTS AND METHODS: The files of all patients sensitized to resorcinol were studied from 1992 to 1999. Over a 20-month period, 983 consecutive patients underwent our standard patch tests where we included resorcinol (1 p. 100 in petrolatum). Co-sensitization with other pro-haptens was studied in all sensitized patients. RESULTS: Sensitization to resorcinol was observed in 0.5 p. 100 of the 983 consecutively tested patients. Contact sensitization was found in 24 patients, who all but one had previously used the anti-wart ointment containing resorcinol. All developed contact eczema on the site of application of the ointment, with generalized urticaria (4 cases), pompholyx (1 case), and generalized papulo-vesicular rash with pompholyx (6 cases). Positive patch tests were observed with resorcinol monobenzoate (15/19 cases), pyrocatechol (7/14 cases), pyrogallol (9/19 cases), salicylaldehyde (2/17 cases), and hydroquinone (6/17 cases). Negative patch tests were observed with phenysalicylate, hexylresorcinol, or fluoresceine. CONCLUSION: Resorcinol should not be applied in high concentrations as it can provoke generalized sensitization and co-sensitization with other pro-haptens.

Administration, Cutaneous↗

[On the enzymatics of the microbial breakdown of pyrazone (author's transl)].

From samples of earth taken in different parts of the world bacteria were isolated which grow on pyrazone as the only source of carbon. When these bacteria are grown in a pyrazone mineral salt medium, four compounds are excreted into the medium. The structures of these compounds furnish information on the catabolic route of pyrazone. Since the suggested scheme of breakdown was incomplete, enzymatic tests were carried out to clarify the matter. It was possible to carry out the first steps of breakdown also in the cell-free extract of the pyrazone-degrading bacteria. For the second step of pyrazone breakdown, 2 different enzymes of the same catalytic activity were identified. For the oxidative cleavage of the pyrocatechole derivative 2 different enzymes were found: an ortho- and a meta-clearing enzyme. The 2-hydroxy muconic acid decarboxylase was identified as a further enzyme. The importance of this enzyme is discussed in connection with the further breakdown.

Bacteria↗