The inhibition of phospholipase A2 by manoalide and manoalide analogues

Manoalide, a natural product from sponge, displays anti-inflammatory activity in vivo. Previous work has shown that manoalide is also a potent covalent inhibitor of the extracellular phospholipase A2 from cobra venom and that the inhibition correlated with a pH-dependent change in manoalide (Lombardo and Dennis (1985) J. Biol. Chem. 260, 7234-7240). Manoalide contains two rings and the opening of either would produce an alpha,beta-unsaturated aldehyde. The cobra venom phospholipase A2 may be able to catalyze the opening or isomerization of one of these rings, raising the possibility that manoalide is acting as a suicide substrate. To ascertain the role of the gamma-lactone ring in the inhibition, we have now investigated a synthetic manoalide analogue, 3(cis,cis-7,10)-hexadecadienyl-4-hydroxy-2-butenolide (HDHB) which contains only the alpha,beta-unsaturated gamma-lactone ring. We have found that the closed and open forms are in rapid equilibrium between pH 4 and 9 with the cyclic form being preferred at acidic pH values and the open cis form preferred at pH 9.5. When the pH is raised above 12, the alpha,beta double bond isomerizes to form trans-HDHB. Once the trans compound is formed, it is stable at all pH values and does not recyclize to the gamma-lactone ring. The observed pKa of 7.7 found for the inhibition of manoalide agrees well with the transition of the closed to the cis form of the gamma-lactone ring. Kinetic experiments with the HDHB compound show that under conditions in which the cis and closed form of the inhibitor are present in equal molar ratios, HDHB is not an irreversible inhibitor, but reversibly competes with substrate. However, the kinetics of this inhibition are complex and do not follow either pure competitive or non-competitive inhibition. The trans-HDHB exhibits similar complex kinetic but is several times more potent. The distinct differences between the behavior of manoalide and HDHB clearly indicate that while the gamma-lactone ring may play an important role in manoalide inhibition, it alone does not produce irreversible inhibition.     

A cross-linked complex between horse pancreatic lipase and colipase

The water soluble carbodiimide N-cyclohexyl-N'-2-morpholinoethyl-carbodiimide-methyl-p-toluolsulfona te was found to effectively covalently cross-link pancreatic colipase to lipase as evidenced by Western blotting experiments using antibodies directed either against lipase or colipase. Moreover the resulting covalent complex has a Mr consistent with a stoichiometry of 1 mol colipase per mol lipase. Cross-linked lipase and colipase retain their activity implying a correct covalent binding between the two proteins. The specificity of the lipase-colipase binding was further supported by the very low amount of cross-linked products when lipase or colipase alone were incubated in the presence of carbodiimide. The formation of a covalent lipase-colipase complex in the presence of carbodiimide clearly demonstrates that the binding between both proteins involves ion pairing. Furthermore, the formation of an active covalent complex strongly suggests that the lipase-colipase binding site is distinct from the colipase interfacial recognition site as well as from the lipase catalytic site.     

Specificity and mechanism of influence of amino acid residues on hepatic clearance of oligopeptides

We have investigated the influence of amino acid residues on hepatic clearance of oligopeptides by determining the rate of disappearance (nmol.(min.g liver)-1) of selective oligopeptides from the medium during isolated rat liver perfusion. (a) N terminus: the rate of disappearance of Ala-Leu was greater (p less than 0.01) than those of Gly-Leu, Phe-Leu, and Arg-Leu (208 +/- 13, 135 +/- 13, 116 +/- 12, and 127 +/- 12, respectively). (b) C terminus: the rate of disappearance of Leu-Ala (244 +/- 18) was significantly greater (p less than 0.01) than that of Leu-Gly (145 +/- 16). (c) Number of residues: with each increase in the number of alanine residues (2-4) there was a significant increase in the rate of peptide disappearance, and conversely, with each increase in the number of glycine residues (2-6) there was a significant decrease in the rate of peptide disappearance. Further studies showed no peptide transport by isolated liver plasma membrane vesicles and no significant correlation between the rates of peptide disappearance and hydrolase activities of the perfusion medium but highly significant correlation with hydrolase activity of plasma membrane. We conclude that certain amino acid residues, such as alanine, enhance hepatic clearance of oligopeptides by increasing their affinity as substrates for plasma membrane peptide hydrolases.     

Ring chromosome 11. A case report and review of the literature

A female infant with severe growth-weight retardation and with a ring chromosome 11, associated with trisomy X in 15% of metaphases, has been reported. A literature review of cases of r(11) shows that the clinical features of these patients, although showing different frequencies, are similar to those of the del(11q) syndrome. It has been suggested that the variability of the mental retardation in r(11) patients is attributable to the unstability of the ring and to the different break points in these two chromosomal rearrangements. The origin of the r(11) was also addressed by studying fragile sites of the parents at 11p15 and 11q25.     

Activation of rat liver glucocorticoid receptor bound to the antiglucocorticoid RU38486

The kinetics of steroid binding to rat liver glucocorticoid receptor (GR) and receptor denaturation were dependent upon the nature of the molecule occupying GR. Both the agonist [triamcinolone acetonide (TA)] and the antagonist (Ru38486) however competed for the same saturable binding site. Despite opposing physiological action, both steroid analogues permitted receptor activation as evident by binding to DNA-cellulose and 9S to 4S shift on sucrose gradient sedimentation. It therefore seems necessary to reevaluate a current notion that antagonist action of RU38486 in rat liver is a result of impaired receptor activation.     

Observations sur la spécificité de la coloration aux acides phosphotungstique et phosphomolybdique

Résumé Les Auteurs ont démontré que, en conditions histochimiques sur du matériel fixé au formol, les acides phosphotungstique et phosphomolybdique ont une double action: 1. une action oxydative à la charge des radicaux PAS-positifs (vic-glycols et éthyleniques) en milieu aqueux, et à la charge des radicaux aminiques en milieu anhydre; cette action entraine la transformation de ces radicaux en aldéhydes; 2. la formation d'une liaison chimique entre les aldéhydes et les molécules des acides phosphomolybdique et phosphotungstique. — La coloration du collagène, au contraire, n'est pas due ni aux radicaux aminiques ni vic-glycols et implique un mécanisme encore inconnu.

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Observations about the specificity of staining of phosphotungstic and phosphomolybdic acid

Summary The authors demonstrated that phosphotungstic and phosphomolybdic acid applied to formol-fixed tissues explete two following actions: 1. An oxidative action on the PAS-positive radicals (double bonds, vic-glycols groups) if in water solution and on aminogroups if in anhydrous solution. This oxidation results in a production of aldehydic radicals 2. The formation of chemical bonds between the aldehydic new-formed groups and the molecules of phosphotungstic or phosphomolybdicacid.— However, the above mentioned mechanism of action is not applicable to the collagen staining for which a different explanation has to be supposed.

     

Cytoplasmic inheritance and ultrastructure of the male generative cell of higher plants

Summary The ultrastructure of the generative cell of the pollen grain has been studied in two different plants: Mirabilis jalapa L., where variegation is transmitted only by the egg cell, and Pelargonium zonale Ait., where variegation can be transmitted also by the pollen grain. It was found that only in Pelargonium zonale Ait. does the male generative cell possesses a great number of proplastids.     

Kinetics of tert-[35S]Butylbicyclophosphorothionate Binding in the Cerebral Cortex of Newborn and Adult Rats: Effects of GABA and Receptor Desensitization

Abstract: The effects of GABA on the kinetics of tert -[³⁵S]butylbicyclophosphorothionate ([³⁵S]TBPS) binding to the convulsant site of GABA_A receptors were studied in membrane suspensions from the cerebral cortex of newborn (1-day-old) and adult (90-day-old) rats. TBPS dissociation was biphasic in neonates and adults, indicating that more than one interconvertible state of [³⁵S]TBPS binding sites may be present in the cerebral cortex. In the absence of GABA, the fast ( t _1/2, 11 min) and slow ( t _1/2, 77 min) components of TBPS dissociation in newborn rats were approximately fourfold slower than in adults. The acceleration of the dissociation rates caused by 2 µ M GABA, however, was more robust in neonates than in adults (six- to ninefold vs. twofold increase, respectively). Moreover, the dissociation rates of TBPS in membranes preincubated with 2 µ M GABA (dissociation started by adding 40 µ M picrotoxin) were two- to fourfold slower than in membranes preincubated without GABA (dissociation started by adding 40 µ M picrotoxin plus 2 µ M GABA). Taken together, these results suggest that (1) the closed state of GABA_A receptors is associated with a more effective steric barrier for the binding of TBPS in neonates compared with adults, (2) GABA produces a larger acceleration of the binding kinetics of TBPS in neonates than in adults, and (3) long incubations with GABA may cause receptor desensitization, which in turn slows down the dissociation rates of TBPS.     

Characterization of the oligosaccharide moiety of VIP receptor from the human pancreatic cell line BxPC-3

The human pancreatic cell line BxPC-3 displays two classes of binding sites with high and low affinity for VIP. The order of potency of VIP-related peptides in inhibiting either [¹²⁵I]VIP or [¹²⁵I]N-AcPACAP27 binding and in stimulating cAMP production was typical of the human VIP receptor. By combining affinity labeling with glycosidase treatments, we have characterized the VIP receptor as a M_r = 68,200 glycoprotein, consisting of a M_r = 39,300 polypeptide core with at least three N-linked oligosaccharide chains. In addition, our results revealed the presence of a low amount of sialic acid residues in the carbohydrate moiety of receptor.