A glutaraldehyde-fuchsin reagent

Synopsis A glutaraldehyde-fuchsin reagent is described. Its preparation avoids the time-consuming ripening period, associated with Gomori's aldehyde-fuchsin. Elastic tissue, insular -cells (after an appropriate prior oxidation) and various other tissue components (e.g. pituitary basophils and acid mucosubstances) can be stained with the reagent 1–2 hours after its preparation.     

The facile HPLC enantioresolution of amino acids,peptides on naphthylethylcarbamate-β-cyclodextrin bonded phases using the acetonitrile-based mobile phase after their pre-column derivatization with phenyl isothiocyanate: factors that affect the resolution

Summary. A variety of -amino acids are enantioresolved for the first time on naphthylethylcarbamate--cyclodextrin bonded phases (i.e., SN- and RN--CD) using the acetonitrile-based mobile phase after their pre-column derivatization with phenyl isothiocyanate in alkaline medium. The resolution is better obtained on RN--CD phase and fails to reproduce if the amino acid is N-benzoylated or N-carbobenzyloxylated under the same chromatographic conditions. The enhanced resolution is believed to be due to the re-location of the hydrogen receptor site from sulfur to nitrogen on the isothiocyanyl fragment of derivatizing reagent, which in turn changes the enantioselectivity. Also, the sulfur atom is larger in size and subject to steric hindrance more significantly in comparison with oxygen. The carboxyl group of amino acid is essential toward a satisfactory resolution. The position of the amino group on the backbone affects the resolution as well. Finally, the resolution is either not observed or unsatisfactory in the reversed- or normal phase mode for most of the amino acids examined in this study.     

Chemical cross-linking of protein to RNA within intact ribosomal subunits from Escherichia coli.

Summary Bifunctional reagents, namely bis-(2-chloroethyl)-amine (nitrogen mustard) and activated esters of 3-(2-bromo-3-oxobutane-1-sulphonyl)-propionic acid (bromo-ketone reagent) are used to cross-link protein to RNA within intact ribosomal subunits. The cross-linked proteins are analysed on two different two-dimensional gel electrophoresis systems, and the existence of a stable cross-linkage is demonstrated by isolating cross-linked protein-oligonucleotide complexes from subunits containing ³²P-labelled RNA. Proteins S3, S4, S5, S9/S11 and S13 from the 30S subunit, and proteins L1 and L2 from the 50S subunit were cross-linked to RNA by the nitrogen mustard, together with a number of other so far unresolved proteins. Correspondingly S3, S4, S7, S9/S11, and L2 were cross-linked by the bromoketone reagent, although in lower yield. The reagents should prove useful for topographical studies on ribosomal subunits, and arguments are presented favouring the use of non-cleavable and relatively non-specific RNA-protein cross-linking reagents for such studies.     

Side effects of reaction media in histochemical blocking procedures

Synopsis 0.2 N NaOH, the reaction medium for 1,2-cyclohexanedione, a specific reagent for arginyl residues in proteins, was found to intensify, at some sites in rat abdominal skin and human gingiva, the Sakaguchi reaction, staining with Pauly's reagent, and anionic dye binding at pH 6.4; at other sites these reactions were reduced, presumably due to extraction of material from sections. 0.2 N NaOH slightly reduced staining after the ninhydrin-Schiff procedure at all sites in rat skin. The interpretation of this finding is obscure, because some sites giving a positive Sakaguchi reaction and staining with anionic dyes failed to stain after the ninhydrin-Schiff procedure. There were also alterations in staining, with the cationic dyes Alcian Blue and Alcian Yellow. It is suggested that 0.2 N NaOH ruptures linkages between polycationic residues of proteins and polanions, demonstrable by Alican Blue. The blockade produced by acetic anhydride-pyridine (4060 v/v) mixtures was stable, in the alkaline conditions required for staining with Pauly's reagent. Pretreatment with pyridine alone reduced tissue binding of anionic dyes.     

The role of light in nitrite reduction; Studies with leaf discs

Summary The reduction of nitrite by leaf discs has been studied. In short term experiments the reduction is markedly stimulated by light, but is not affected by the absence of oxygen or carbon dioxide from the gas phase. Carbon dioxide assimilation is more sensitive than nitrite reduction to 3-(3,4-dichloro-)-1,1-dimethyl urea (DCMU) inhibition. Uncouplers such as carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) do not inhibit nitrite reduction although dinitrophenol (DNP) has a small effect.Although nitrite stimulates oxygen evolution in the light in the absence of CO₂ the stoichiometry of nitrite reduction to oxygen evolution is much less than would be predicted if nitrite is simply acting as a classical Hill reagent.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - DNP 2,4-dinitrophenol - CCCP carbonyl cyanide m-chlorophenylhydrazone     

Basic carboxyl groups of hemoglobin S: Influence of oxy-deoxy conformation on the chemical reactivity of Glu-43(β)

The -carboxyl groups of Glu-43() and Glu-22() of hemoglobin-S (HbS), two intermolecular contact residues of deoxy protein, are activated by carbodiimide atp H 6.0. The selectivity of the modification by the two nucleophiles, glycine ethyl ester (GEE) and glucosamine, is distinct. Influence ofN-hydroxysulfosuccinimide, a reagent that rescues carbodiimide-activated carboxyl (O-acyl isourea) as sulfo-NHS ester, on the overall selectivity and efficiency of the coupling of Glu-22() and Glu-43() with nucleophiles has been investigated. Sulfo-NHS increases the extent of coupling of nucleophiles to HbS. The rescuing efficiency of sulfo-NHS(increase in modification) with GEE and galactosamine as nucleophiles is 2.0 and 2.8, respectively. In the presence of sulfo-NHS, the extent of modification of a carboxyl group is a direct reflection of the extent to which it is activated (i.e., the protonation state of the carboxyl group). The modification reaction exhibits very high selectivity for Glu-43() with GEE and galactosamine (GA) in the presence of sulfo-NHS. From the studies of the kinetics of amidation of oxy-HbS at its Glu-43() (i.e., chemical reactivity) as a function of thepH in the region of 5.5–7.5, the apparentpKa of its -carboxyl group has been calculated to be 6.35. Deoxygenation of HbS, nearly doubles the chemical reactivity of Glu-43() of HbS atpH 7.0. It is suggested that the increased hydrophobicity of the microenvironment of Glu-43(), which occurs on deoxygenation of the protein, is reflected as the increased chemical reactivity of the -carboxyl group and could be one of the crucial preludes to the polymerization process.     

Properties of phosphorylated NADP+-specific glutamate dehydrogenase fromEscherichia coli

The NADP⁺-specific glutamate dehydrogenase (GDH) fromEscherichia coli strain D₅H₃G₇, an enzyme that catalyzes the interconversion of -ketoglutarate andl-glutamate, has been shown to be phosphorylated in vitro in an ATP-dependent enzymatic reaction. The phosphorylated protein is extremely acid labile and is unstable at high pH. Treatment of GDH with diethyl pyrocarbonate (DEP), a histidine-modifying reagent, blocked the incorporation of³²P from [-³²P]ATP. GDH catalytic activity was also inhibited by DEP treatment. Hydroxylamine, a reagent hydrolyzing phosphoramidates, catalyzed the removal of phosphate from phosphorylated GDH, suggesting that GDH may be phosphorylated at a histidine residue(s). A total enzymatic hydrolysis of phosphorylated GDH, which was electroeluted from a native polyacrylamide gel, was analyzed by a Dowex 1-8X anion exchange chromatography. The presence of³²P-labeled 3-phosphohistidine, characterized and identified from this hydrolysate, demonstrates that a histidine residue(s) is the site of phosphorylation.     

High Efficiency Gene Transfer by Multiple Transfection Protocol

A high efficiency transfection protocol employing a common polycationic lipid is described. Using LipofectAMINE, a widely used transfection reagent, we transfected 293T cells with a plasmid harboring the -galactosidase (-gal) gene. The transfection efficiency was determined by direct staining for X-gal. The conventional transfection protocol achieved an efficiency of <40% while our protocol, which employs the repetition of transfection a few times, achieved an efficiency of approximately 80%. Thus, a dramatic increase in transfection efficiency can be obtained by simply repeating transfection with the use of a common polycationic lipid. This method will be useful in many molecular biological experiments.     

Remarks on the detection of osmium derivatives in tissue sections

Summary The reaction of substances with osmium tetroxide in tissue sections is evident when primary or secondary blackening occurs. Many substances are not colored, however, despite the fact that they may react with osmium tetroxide. Osmium derivatives bound in such structures may be either in an insufficient amount or in a higher valency state or both. For the demonstration of such weakly colored bound osmium derivatives some chemical reagents including those used in analytical chemistry were tested. Not all reagents used were equally effective. Tschugajev's reagent with thiourea is unreliable for in situ detection in tissue sections because the resulting colored complex is soluble and escapes from the section into solution. Yellow ammonium sulphide is not sensitive enough. N-phenyl-p-phenylene diamine, N-dimethyl-p-phenylene diamine, benzidine, o-dianisidine, N-diethyl-p-phenylene diamine sulphate, p-aminophenol, p-phenylene diamine, N-methyl-p-aminophenol sulphate, 2,4-diaminophenol hydrochloride, -naphthylamine, -naphthylamine, diphenylcarbazone, 8-amino-1-naphthol-3,6-disulphonic acid (H acid), aniline, tannin, proved to be the most suitable detecting reagents. By these reagents large quantities of osmium pigments could be demonstrated in many structures even after the most efficient lipid extraction methods.     

Solid Phase Synthesis of Phosphorothioate Oligonucleotides Utilizing Diethyldithiocarbonate Disulfide (DDD) as an Efficient Sulfur Transfer Reagent

Abstract

Diethyldithiodicarbonate (DDD), a cheap and easily prepared compound, is found to be a rapid and efficient sulfurizing reagent in solid phase synthesis of phosphorothioate oligodeoxyribonucleotides via the phosphoramidite approach. Product yield and quality based on IP-LC-MS compares well with high quality oligonucleotides synthesized using phenylacetyl disulfide (PADS) which is being used for manufacture of our antisense drugs.      

Isolation and culture of cotton ovule epidermal protoplasts (prefiber cells) and analysis of the regenerated wall

Culture protocols were developed and characterization of the regenerated cell walls was performed for protoplasts of cotton (Gossypium hirsutum L., L., var. Acala SJ-2) ovule epidermal cells. This work was undertaken in order to extend studies concerning nutritional effects and regulation of nucleotide sugar incorporation into -1,3- and -1,4-glucan components of cotton fiber cell walls. Protein and carbohydrate polymers and recovered from the culture medium. Analysis of a cellular fraction indicated that the majority of ¹⁴C incorporated from [¹⁴C] glucose was present in the hot-water-soluble fraction of the cells. The majority of label incorporated into cell wall material could be solubilized with acetic-nitric reagent, indicative of noncellulosic material, and characterized as -1,3-linked glucans. Only 5 to 15% of the regenerated cell wall could be characterized as -1,4-linked glucose indicative of cellulose.     

Improvement of oxygen-carrying properties of human hemoglobin by chemical modification with a benzene hexacarboxylate-monosubstituted polyoxyethylene

Benzene hexacarboxylate-monosubstituted polyoxyethylene on contact with Hb decreases its oxygen affinity, probably because it specifically interacts with the amino groups of the phosphate-binding site. This site specificity was used to direct the covalent coupling of this polymeric reagent with hemoglobin, in the vicinity of this cleft in order to obtain conjugates with low oxygen affinity and well-defined molecular weight. Such conjugates could thus be regarded as potential candidates for blood substitutes. Covalent fixation of this polymeric site-labeling reagent onto hemoglobin was carried out with the oxy and the deoxy form in the presence of a water soluble carbodiimide. It turns out that the oxygen-binding properties of the resulting hemoglobin derivatives depend on the reaction conditions, yet in all cases the oxygen affinity of the modified protein was lower than that of native hemoglobin and was no longer affected by organic phosphates. These results indicate that phosphate-binding site amines are probably involved in the covalent coupling, although in some conjugates (especially those prepared with high ratios of reagents) other amino groups participate also in the linking to the polymer. Chromatographic analysis and trypic peptide mapping of some conjugates evidenced that the -terminal valine residue was in fact the preferential binding site of hexacarboxylate-monosubstituted polyoxyethylene.     

The Modular Organization of Multifunctional Peptide Synthetases

Gramicidin S synthetase 2 from B. brevis was affinity labeled at its valine thiolation center with the thiol reagent N-[³H]ethylmaleimide. From a tryptic digest of the enzyme–inhibitor complex a radioactive fragment was isolated in pure form by two reversed-phase HPLC steps. It was identified by liquid-phase N-terminal sequencing in combination with electrospray mass spectrometry (ESI-MS) as a hexadecapeptide containing the thiolation motif LGG(H/D)S(L/I). By ESI-MS it was demonstrated that a 4-phosphopantetheine cofactor was attached to this fragment at its reactive serine. These results are consistent with the Multiple Carrier Model of nonribosomal peptide biosynthesis. Site-specific mutagenesis has been performed in thiolation, elongation, and epimerization motifs of some of the modules of surfactin synthetase from B. subtilis to clarify the function of prominent conserved amino acid residues in the intermediate steps of peptide biosynthesis. The modular structure of multifunctional peptide synthetases is discussed.     

The use of diaminobenzidine (DAB) for the histochemical demonstration of cytochrome oxidase activity in unfixed plant tissues

Summary Cytochrome oxidase activity was demonstrated in unfixed root segments from Lupinus albus at the ultrastructural level using the osmiophilic reagent 3,3-diaminobenzidine (DAB). Precipitate, the formation of which was completely inhibited by 0.01 M KCN, and observed almost entirely on mitochondrial cristae, is considered to be produced by cytochrome oxidase activity. Heterogeneity of mitochondria as to the intensity of the reaction in the same cell could not be established with certainity. However, mitochondria of the root tip cells and cells belonging to the plerome consistently did not show histochemically demonstrable cytochrome oxidase activity.     

Histochemical demonstration of β-glucuronidase activity during sequential molar development in the Swiss albino mouse

Summary Using naphthol AS-BI -D glucuronide as a substrate and diazotized pararosanilin as a coupling reagent, the distribution of -glucuronidase was studied throughout mouse molar tooth development. A strongly positive reaction was not only observed in some of the cellular components of the stellate reticulum of the enamel organ, but also in certain cells of the subodontoblastic cell zone. A moderate reaction was noted in the distal cytoplasm of the odontoblasts particularly in the older ones which were found in the cuspal areas. A mild reaction was elicited in the stratum intermedium and a weak one in the ameloblasts and prospective dental pulp.Supported PHS. Grant No. 2800-02 National Institute of Dental Research. National Institutes of Health.     

Evidence for a Conformational Change in Subunit III of Bovine Heart Mitochondrial Cytochrome c Oxidase1

The role of subunit III in the function of mitochondrial cytochrome c oxidase is not clearly understood. Previous work has shown that chemical modification of subunit III with N,N-dicyclohexylcarbodiimide (DCCD) reduced the proton-pumping efficiency of the enzyme by an unknown mechanism. In the current work, we have employed biochemical approaches to determine if a conformational change is occurring within subunit III after DCCD modification. Control and DCCD modified beef heart enzyme were subjected to limited proteolysis in nondenaturing detergent solution. Subunit III in DCCD treated enzyme was more susceptible to chymotrypsin digestion than subunit III in the control enzyme. We also labeled control and DCCD-modified enzyme with iodoacetyl—biotin, a sulfhydryl reagent, and found that subunit III of the DCCD-modified enzyme was more reactive when compared to subunit III of the control enzyme, indicating an increase in reactivity of subunit III upon DCCD binding. The cross linking of subunit III of the enzyme induced by the heterobifunctional reagent, N-succinimidyl(4-azidophenyl -1,3-dithio)-propionate (SADP), was inhibited by DCCD modification, suggesting that DCCD binding prevents the intersubunit cross linking of subunit III. Our results suggest that DCCD modification of subunit III causes a conformational change, which most likely disrupts critical hydrogen bonds within the subunit and also those at the interface between subunits III and I in the enzyme. The conformational change induced in subunit III by covalent DCCD binding is the most likely mechanism for the previously observed inhibition of proton-pumping activity.     

High-performance liquid chromatographic resolution of (R,S)-α-alkyl-α-amino acids as diastereomeric derivatives

Summary A number (27) of racemic-alkyl--amino acids (AAA) were derivatized either witho-phthaldialdehyde (OPA) in combination withN-t-butoxycarbonyl-L-cysteine (Boc-Cys) orN-acetyl-L-cysteine (Ac-Cys), or withN ²-(5-fluoro-2,4-dinitrophenyl)-L-alanine amide (Marfey's reagent). The resolution of the diastereoisomers formed was investigated by reversed-phase (C₁₈) high-performance liquid chromatography (HPLC) using gradient elution conditions employing sodium phosphate buffers of pH 7.2 together with acetonitrile, and fluorescence detection at 344 nm (excitation) and 443 nm (emission) for the OPA/Boc-Cys or OPA/Ac-Cys derivatives. For the diastereomers formed by derivatization with Marfey's reagent triethylammonium phosphate buffers of pH 3.0 (pH 7.2 for acidic AAA) together with acetonitrile, and u.v. detection at 340 nm were used. Whereas with Marfey's reagent all diastereomers of AAA showed complete, or almost complete, resolution, only 8, or 11, respectively of the diastereomers formed by derivatization with OPA/Boc-Cys or OPA/Ac-Cys were resolved under the chromatographic conditions used.     

Characterization of the guanosine 3′∶5′-monophosphate-dependent protein kinase from silkworm eggs and analysis of the endogenous protein substrates

Summary In a previous paper, two types of cyclic nucleotide-dependent protein kinases, namely cGMP dependent G-kinase and cAMP dependent A-kinase, in silkworm eggs has been reported (Takahashi et al. 1975; Takahashi 1976). One of these, G-kinase, has now been purified 2400-fold by means of ammonium sulfate fractionation, chromatography on hydroxylapatite, DEAE cellulose, and gel filtration.Some of the properties of the enzyme are described. The enzyme is highly dependent on cGMP; it is strongly inhibited by GTP in a noncompetitive manner not only for ATP but also for cGMP. GTP was found to be highly inhibitory on G-kinases from various tissues of the silkworm, but did not inhibit the A-kinase.Incubation of the egg extract with [-³²P]ATP and Mg²⁺ led to the formation of three major³²P-labelled proteins, with molecular weights of 42.000, 70.000 and 180.000 as analyzed by SDS polyacrylamide gel electrophoresis. Two of them corresponded to the subunits of vitellin.The silkworm vitellin was effectively phosphorylated both by the highly purified G-kinase and by the A-kinase. It is concluded that the G-kinase is involved in the phosphorylation of vitellin in developing silkworm eggs.Abbreviations cAMP adenosine 35-monophosphate - cGMP guanosine 35-monophosphate - A-kinase adenosine 35-monophosphate-dependent protein kinase - G-kinase guanosine 35-monophosphate-dependent protein kinase - MIX 1-methyl-3-isobutylxanthine     

Peptide modification by incorporation ofα-trifluoromethyl substituted amino acids

Summary Metabolic stabilization of pharmacologically active peptides can be achieved by incorporation of sterically hindered non-natural amino acids, e.g. C ^, -disubstituted amino acids.-Trifluoromethyl substituted amino acids, a subclass of C ^, -disubstituted amino acids, also fulfil this requirement while featuring additional properties based on the electronic influence of the fluorine substituents.This review summarizes the results concerning the stability of peptides containing-TFM amino acids towards proteolysis by-chymotrypsin. Furthermore, configurational effects of-TFMAla on the proteolytic stability of peptides are explained using empirical force field calculations. The influence of-TFMAla incorporation on the secondary structure of selected tripeptide amides is compared to the effects exerted by its fluorine-free analogue, aminoisobutyric acid.Finally, results on metabolic stabilization and biological activity of modified thyrotropin releasing hormone are interpreted.     

Contextualizing Images in Dreams and Daydreams

A contextualizing image (CI) is a powerful central image of a dream which appears to contextualize (provide a picture-context for) the dreamer's emotion. For instance, dreamers who have experienced any serious traumatic event sometimes dream, I was overwhelmed by a tidal wave. This appears to picture their feeling of terror and/or vulnerability.A scoring system for CIs is examined here and is applied to dreams and daydreams supplied by 40 students. Two raters scoring dreams on a blind basis showed good inter-rater reliability. Recent dreams were shown to have more as well as more intense CIs than recent daydreams; likewise, dreams that stand out had more intense CIs than daydreams that stand out. Students with thin boundaries had more and more intense CIs than students with thick boundaries in their recent dreams and nightmare, but not so clearly in dreams and nightmares that stand out. The emotions judged as contextualized by the powerful images tended towards fear/terror and helplessness/vulnerability in dreams (especially in dreams that stand out) whereas emotions contextualized by images in daydreams showed a wide range with no clusters.