Inositol 1,4,5-trisphosphate (IP3) induced rapid formation of thromboxane B2 in saponin-permeabilised human platelets: mechanism of IP3 action

The mechanism of IP3-induced activation of saponin-permeabilised platelets has been examined. Saponin permeabilization resulted in the leakage of low-Mr substances into and from the cells without loss of cytoplasmic proteins. Addition of IP3 rapidly induced a dose-related formation of thromboxane B2 and release into the medium, leading to the responses of shape change, aggregation and [14C]5HT release. These responses were inhibited by the thromboxane A2 receptor antagonist AH23848. The IP3-induced release of 45Ca from intracellular stores was not affected by indomethacin. Synthesis of thromboxane was inhibited if Ca2+ elevation was prevented by using Ca-EGTA buffers during permeabilization. These studies indicate that IP3-induced activation was due to Ca2+ mobilisation leading to phospholipase activation and thromboxane synthesis.     

The HsdS polypeptide of the Type IC restriction enzyme Eco R124 is a sequence-specific DNA-binding protein

The HsdS and HsdM polypeptides of the type IC restriction enzyme EcoR124 have been purified independently and used in a set of gel retardation experiments to determine the minimum requirements for sequence-specific recognition of DNA by this enzyme. The HsdS polypeptide alone is able to bind to DNA in a sequence-specific manner. In addition, whilst the presence of the HsdM polypeptide gives rise to a stimulation of DNA binding by the HsdS subunit it is not clear whether, under the conditions of the experiments reported here, the HsdS subunit maintains the same interactions with the HsdM subunits observed in the absence of DNA.     

Anatomical evidence for interaction of ACTH1–39 immunostained fibers and hypothalamic paraventricular neurons that project to the dorsal vagal complex

Summary Anatomical evidence is presented for an interaction of ACTH^1–39 immunostained fibers and a specific population of hypothalamic paraventricular (PVN) neurons; these neurons project to the dorsal vagal complex (DVC) of brainstem medulla. Bilateral injection of 10% HRP-WGA into DVC is incorporated into nerve terminals and transported retrogradely to cell bodies in the parvocellular subdivision of PVN, as revealed by standard HRP-WGA histochemistry or antibody to wheatgerm agglutinin followed by immunocytochemical techniques. Labeled cells are localized predominantly in the ventral portion of the caudal medial parvocellular subdivision and ventrolaterally in the posterior subnucleus of PVN. Few labeled cells are seen in the anterior parvocellular PVN, rostrally in the medial parvocellular component and in the dorsal cap. HRP-WGA cells are rarely observed in the magnocellular divisions of PVN. Dual-staining immunocytochemical-retrograde tracing techniques in the same tissue section demonstrate ACTH^1–39 fibers in intimate anatomical proximity to parvocellular PVN neurons that project to DVC. It is suggested that this interaction may partially account for the known cardiovascular effects of opiocorins and supports the role of the paraventricular nucleus in hypothalamie integration and modulation of cardiovascular control.     

The preparation and properties of ficin chemically attached to carboxymethylcellulose

1. Purified ficin has been coupled to four CM-celluloses by reaction with their acid azide derivatives. Insoluble products containing 1.8-4.7mg. of ficin/100mg. of product and retaining 8.0-12.0% of the free enzyme's esterase activity have been obtained. 2. The amount of bound ficin in these preparations is dependent on the degree of carboxymethyl substitution of the CM-cellulose to which the ficin is attached. 3. A shift of the alkaline limb of the pH-activity curve of ficin when chemically attached to CM-cellulose has been shown. 4. Only a small loss has been observed in the enzymic activity of these products when stored at 2 degrees for 4 months. They are more resistant than free enzyme to heat denaturation. 5. Columns of CM-cellulose-ficin have been packed. The degree of hydrolysis of perfused substrate has been measured for different flow rates through the column. 6. The properties of these derivatives have been discussed.     

The kinetic mechanism of ox liver glutamate dehydrogenase in the presence of the allosteric effector ADP. The oxidative deamination of L-glutamate.

In steady-state kinetic studies of ox liver glutamate dehydrogenase in 0.11 M-potassium phosphate buffer, pH7, at 25 degrees C, the concentration of ADP was varied from 0.5 to 1000 microM. Inhibition was observed except when the concentrations of both glutamate and coenzyme were high, when activation was seen. With NAD+ or NADP+ as coenzyme, 200 microM-ADP was sufficient to saturate the enzyme with respect to the major effect of this nucleotide. In the presence of 210 microM-ADP, widely varied concentrations of coenzyme give linear Lineweaver-Burk plots, in marked contrast with results obtained previously for kinetics without ADP. This has allowed evaluation for the reaction with NAD+, NADP+ and acetylpyridine-adenine dinucleotide (315 microM-ADP in the last case) of all four initial rate parameters, i.e. the phi coefficients in the equation: (Formula: see text) where A is coenzyme and B is glutamate. The relative constancy of phi B and of phi AB/phi A with the different coenzymes point to a compulsory-order mechanism with glutamate as the leading substrate. This conclusion, though unexpected, agrees well with various previous observations on the binding of oxidized coenzyme.     

Equilibrium folding of dimeric class mu glutathione transferases involves a stable monomeric intermediate

The conformational stabilities of two homodimeric class mu glutathione transferases (GSTM1-1 and GSTM2-2) were studied by urea- and guanidinium chloride-induced denaturation. Unfolding is reversible and structural changes were followed with far-ultraviolet circular dichroism, tryptophan fluorescence, enzyme activity, chemical cross-linking, and size-exclusion chromatography. Disruption of secondary structure occurs as a monophasic transition and is independent of protein concentration. Changes in tertiary structure occur as two transitions; the first is protein concentration dependent, while the second is weakly dependent (GSTM1-1) or independent (GSTM2-2). The second transition corresponds with the secondary structure transition. Loss in catalytic activity occurs as two transitions for GSTM1-1 and as one transition for GSTM2-2. These transitions are dependent upon protein concentration. The first deactivation transition coincides with the first tertiary structure transition. Dimer dissociation occurs prior to disruption of secondary structure. The data suggest that the equilibrium unfolding/refolding of the class mu glutathione transferases M1-1 and M2-2 proceed via a three-state process: N(2) <--> 2I <--> 2U. Although GSTM1-1 and GSTM2-2 are homologous (78% identity/94% homology), their N(2) tertiary structures are not identical. Dissociation of the GSTM1-1 dimer to structured monomers (I) occurs at lower denaturant concentrations than for GSTM2-2. The monomeric intermediate for GSTM1-1 is, however, more stable than the intermediate for GSTM2-2. The intermediates are catalytically inactive and display nativelike secondary structure. Guanidinium chloride-induced denaturation yields monomeric intermediates, which have a more loosely packed tertiary structure displaying enhanced solvent exposure of its tryptophans and enhanced ANS binding. The three-state model for the class mu enzymes is in contrast to the equilibrium two-state models previously proposed for representatives of classes alpha/pi/Sj26 GSTs. Class mu subunits appear to be intrinsically more stable than those of the other GST classes.     

Aza-bicyclic amino acid sulfonamides as alpha(4)beta(1)/alpha(4)beta(7) integrin antagonists

The design, synthesis, and biological activity of novel alpha(4)beta(1) and alpha(4)beta(7) integrin antagonists, containing a bridged azabicyclic nucleus, are reported. Conformational analysis of targets containing an azabicyclo[2.2.2]octane carboxylic acid and known integrin antagonists indicated that this azabicycle would be a suitable molecular scaffold. Variation of substituents on the pendant arylsulfonamide and phenylalanine groups resulted in potent alpha(4)beta(1)-selective and dual alpha(4)beta(1)/alpha(4)beta(7) antagonists. Potent compounds 11i, 11h, and 14 were effective in the antigen-sensitized sheep model of asthma.     

Enhancing the catalytic repertoire of nucleic acids: a systematic study of linker length and rigidity

The incorporation of potentially catalytic groups in DNA is of interest for the in vitro selection of novel deoxyribozymes. A series of 10 C5-modified analogues of 2'-deoxyuridine triphosphate have been synthesised that possess side chains of differing flexibility and bearing a primary amino or imidazole functionality. For each series of nucleotide analogues differing degrees of flexibility of the C5 side chain was achieved through the use of alkynyl, alkenyl and alkyl moieties. The imidazole function was conjugated to these C5-amino-modified nucleotides using either imidazole 4-acetic acid or imidazole 4-acrylic acid (urocanic acid). The substrate properties of the nucleotides (fully replacing dTTP) with TAQ polymerase during PCR have been investigated in order to evaluate their potential applications for in vitro selection experiments. 5-(3-Aminopropynyl)dUTP and 5-(E-3-aminopropenyl)dUTP and their imidazole 4-acetic acid- and urocanic acid-modified conjugates were found to be substrates. In contrast, C5-amino-modified dUTPs with alkane or Z-alkene linkers and their corresponding conjugates were not substrates. The incorporation of these analogues during PCR has been confirmed by inhibition of restriction enzyme digestion using XBAI and by mass spectrometry of the PCR products.