Preparation of an N-acetyl-octapeptide of cholecystokinin: The role of N-acetylation in protecting the octapeptide from degradation by smooth muscle tissues

The C-terminal octapeptide of cholecystokinin (CCK-8) was acetylated on its lone N-terminal amino group using acetic anhydride in N,N-dimethylformamide. The acetylated derivative (Ac-CCK-8) and unreacted CCK-8 were separated from acetic anhydride and other reaction products by fractionation on Sephadex LH-20. Final purification was by thin-layer isoelectric focusing in a pH 2.5–4.0 gradient. The immunochemical properties of the octapeptide were unaffected by acetylation as measured by radioimmunoassay. The N-acetylated-octapeptide was equally as effective as unmodified CCK-8 in producing concentratiion-dependen isometric tension development in isolated cat gallbladder strips. Acetylation did, however, protect CCK-8 from N-terminal degradation by soluble peptidases that eluted from gallbladder and other smooth muscle tissues of the cat. Unmodified CCK-8 was degraded rapidly in the presence of these tissues and in buffers previously exposed to the same tissues. In contrast, the Ac-CCK-8 was resistant to N-terminal degradation under the same conditions. Degradation of CCK-8 from its N-terminus produces biologically inactive derivatives and could adversely affect in vitro studies. Since the acetylated-CCK-8 retained full biological and immunological activity, its use would eliminate the effect of extracellular proteolysis on CCK-8 action.     

Effectivity of dolichyl phosphates with different chain lengths as acceptors of nucleotide activated sugars

Chemical synthesis of different S-forms of dolichyl-P was performed in order to investigate the use of these polyprenes in mannosyl, glucosyl and glucosaminyl transferase reactions. Determination of the Vmax values for a series of dolichyl-P demonstrated that the velocities of transferase reactions with all those dolichyl-P derivatives present in animal tissues are largely the same. The apparentK _m values for the various dolichyl-P in the transferase system studied differed, but this property does not appear to have physiological importance.     

Conversion of an apparent 100 kDa folate binding protein from human milk,choroid plexus and semen to a 25 kDa molecular species by phosphatidylinositol-specific phospholipase C

Gel filtration studies in the presence of Triton X-100 showed that treatment with phosphatidylinositol-specific phospholipase C reduced the apparent molecular size of the 100 kDa folate binding protein from human milk, choroid plexus and semen to 25 kDa. Cleavage of a hydrophobic glycosly phosphatidylinositol domain (a membrane anchor) inserting the protein into Triton X-100 micelles could account for this phenomenon.     

Six guaianolides from Stylotrichium rotundifolium

The investigation of Stylotrichium rotundifolium afforded the epoxide of β-sesquiphellandrene, a new derivative of geranylnerol and six guaianolides not isolated previously. Furthermore, two germacranolides also present in a Lasiolaena species and several known compounds were isolated. The structures were elucidated by detailed ¹H NMR investigations. The chemotaxonomic situation is discussed briefly.     

Simultaneous determination of citalopram and tadalafil by the second derivative synchronous fluorescence method in biological fluids; application of Box–Behnken optimization design

This is the first study focusing solely on that determination of tadalafil in the presence of citalopram as an antidepressant drug. The determination in biological fluids of a co‐administered antidepressant drug and a sexual stimulation drug is a very critical and important step for psychotic and ischaemic heart disease patients, especially in cases of emergency and this requires therapeutic drug monitoring. A sensitive, efficient and rapid assay was selected satisfactorily and applied for simultaneous determination of citalopram and tadalafil either in their pure forms, in tablet dosage forms or in spiked human plasma. There was a large overlap for both drugs, forming the broad band found in conventional fluorescence spectra and their related synchronous fluorescence intensity. Therefore, the development of a highly sensitive second derivative synchronous fluorescence method was demonstrated that removed this overlap. The proposed method depended on measuring the amplitudes of the second derivative of synchronous fluorescence intensity at suitable wavelengths of 301 nm and 367 nm for citalopram and tadalafil at Δλ = 60 nm, respectively. Box–Behnken design as a response surface methodology was used to fit models and create an optimization process encompassing a set of factors and resulting in an optimum response value specifically designed for this method. Under optimum conditions, the linear dynamic ranges for citalopram and tadalafil estimation were 20–900 and 5–400 ng ml^?1 with detection limits of 5.40 and 1.43 ng ml^?1, respectively.     

Presence of di- and polyamines covalently bound to protein in rat liver

Acid hydrolysis of trichloroacetic acid precipitate from rat tissue (liver, kidney and testis) homogenate released significant amounts of acid-insoluble putrescine, spermidine and spermine. Following incubation of liver homogenate with [1,4-¹⁴C]putrescine, 1.4% of total radioactivity and 1.0% of labelled diamine were recovered in the acid-insoluble fraction. Exhaustive digestion of acid-precipitable material with proteinases (Pronase, aminopeptidase M, carboxipeptidase A, B and Y) revealed the presence of di- and polyamines and of N¹-(γ-glutamyl)spermidine, N¹-(γ-glutamyl)sperminine and N¹, N¹²-bis(γ-glutamyl)spermine. These derivatives were identified both by chromatographic analysis and by enzymatic digestion with purified γ-glutamylamine cyclotransferase. The finding of di- and polyamine γ-glutamyl derivatives in the proteinase-digested acid-insoluble fraction of homogenate may be considered as a proof of the in vivo transglutaminase-catalyzed binding of polyamines to proteins. This evidence suggests that di- and polyamines might have an important role in mammalian tissues through covalent binding to proteins by either one or both the primary amino groups.     

Nutrient-specific proteomic analysis of the mucin degrading bacterium Akkermansia muciniphila

Akkermansia muciniphila is a prominent mucin-degrading bacterium that acts as a keystone species in regulating the human gut microbiota. Despite recently increasing research into this bacterium and its relevance to human health, a high-resolution database of its functional proteins remains scarce. Here, we provide a proteomic overview of A. muciniphila grown in different nutrient conditions ranging from defined to complex. Of 2318 protein-coding genes in the genome, we identified 841 (40%) that were expressed at the protein level. Overall, proteins involved in energy production and carbohydrate metabolism indicate that A. muciniphila relies mainly on the Embden-Meyerhof-Parnas pathway, and produces short-chain fatty acids through anaerobic fermentation in a nutrient-specific manner. Moreover, this bacterium possesses a broad repertoire of glycosyl hydrolases, together with putative peptidases and sulfatases, to cleave O-glycosylated mucin. Of them, putative mucin-degrading enzymes (Amuc_1220, Amuc_1120, Amuc_0052, Amuc_0480, and Amuc_0060) are highly abundant in the mucin-supplemented media. Furthermore, A. muciniphila uses mucin-derived monosaccharides as sources of energy and cell wall biogenesis. Our dataset provides nutrient-dependent global proteomes of A. muciniphila ATCC BAA-835 to offer insights into its metabolic functions that shape the composition of the human gut microbiota via mucin degradation.