Bacillus subtilis alpha-amylase: interactions of a partially folded conformer with small unilamellar vesicles.
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We studied the interactions between conformers of exocellular alpha-amylase and small unilamellar vesicles (SUV) composed of the major membrane lipids of Bacillus subtilis under physiological conditions of pH, temperature and ionic strength. Using fluorescence spectroscopy, surface plasmon resonance (SPR) and phase separation, we show that the native alpha-amylase has no affinity for the SUV, whereas a partially folded form, displaying structural properties in common with the competent state for secretion, binds to the vesicles (KA approximately 10(5) M(-1)). This association prevented its subsequent folding. The complex was destabilized in the presence of PrsA, a major peripheric lipoprotein of B. subtilis which displays a strong affinity for SUV (KA approximately 1.5x10(8) M(-1)). Vesicles coated with PrsA lost their ability to bind the partially folded conformer. The approach in vitro, in which our aim was to mimic the last stage of alpha-amylase translocation, indicates that PrsA possibly helps, in vivo, the secreted protein to acquire its native conformation by modulating the interaction between the latter and the lipid polar heads on the trans side of the cytoplasmic membrane. (+info)
Behaviour of ejaculated spermatozoa from bull, boar and ram during thin-layer countercurrent partition in aqueous two-phase systems.
(10/131)
Ejaculated spermatozoa from bulls, boars and rams were subjected to thin-layer countercurrent partition in aqueous two-phase systems composed of dextran T500 and polyethylene glycol 6000 (PEG) in sucrose-based Hepes-buffered media. In the basal system, the great majority of spermatozoa tended to partition with the dextran-rich bottom phase; however, by including very low levels of phosphate, they could be made to partition increasingly with the PEG-rich top phase (complete at 10 mM phosphate). A procedure was developed for carrying out four separations simultaneously under identical conditions, whereby it could be shown that distribution varied with the number of spermatozoa in the sample. In the case of bull, the effect of cell number could be reduced considerably by inclusion of small quantities of seminal plasma in the phase system, but no such effect was found for ram or boar. Considerable variation in distribution pattern was seen between samples, which did not appear to be due to technical inconsistency. Livability in the phase systems was also variable, and we believe that PEG may exert a detergent-like effect on the sperm surface that is exacerbated in highly defined media free of protective proteins. (+info)
Preparative scale purification of shidasterone, 2-deoxy-polypodine b and 9a,20-dihydroxyecdysone from Silene italica ssp. nemoralis.
(11/131)
A suitable combination of preparative scale separation methods results in effective clean-up of the ecdysteroids of Silene italica ssp. nemoralis (Waldst. and Kit.) Nyman. The isolation of minor ecdysteroids from the partially purified extract is based on the use of both droplet counter-current chromatography and low-pressure reversed-phase liquid chromatography. The purification is completed by preparative thin-layer chromatography and preparative high-performance liquid chromatography to obtain the minor ecdysteroids, such as 2-deoxy-20-hydroxyecdysone, shidasterone, 2-deoxy-polypodine B, makisterone C, and 9alpha,20-dihydroxyecdysone. (+info)
Counter-current transfer in reproductive biology.
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Heat and substances, including gases, steroids and peptide hormones, can pass from venous blood, interstitial fluid and lymph to the arterial blood; the process is called local counter-current transfer. It has been found in various reproductive organs in many animal species and in man: from the testis to the testis and epididymis; from the ovary to the ovary, tube and tubal corner of the uterus; from the tube and uterus to the ovary; from vagina to uterus; and even between brain blood vessels. Local transfer within the ovary has also been found. Local cooling that creates temperature gradients between organs or within an organ is one aspect of the transfer. Physiologically, the transfer also facilitates local feedback regulation of organ function in a process situated between general distribution of hormones through the systemic circulation and paracrine regulation. Counter-current transfer of drugs after local application opens up new possibilities for treatment. (+info)
Isolation of flavanol-anthocyanin adducts by countercurrent chromatography.
(13/131)
Pigments of the flavanol-anthocyanin (F-A+) type detected earlier in wine are synthesized using a protocol adapted from the synthesis of procyanidin dimers. The F-A+ adduct thus obtained is purified by countercurrent liquid-liquid partition, currently referred to as countercurrent chromatography (CCC). The solvent system consists of tert-butyl methyl ether-n-butanol-acetonitrile-water (2:2:1:5, acidified with 0.1% trifluoroacetyl) with the light organic phase acting as a stationary phase and the aqueous phase as the mobile phase. Four fractions are recovered and analyzed by high-performance liquid chromatography coupled to a diode-array detector and electrospray ionization mass spectrometer. The multilayer CCC method allowed the separation of pigments in three different groups. The first group consists of hydrosoluble pigments present in fraction 1; the second group consists of the F-A+ adducts [catechin-malvidin 3 glucoside (Mv3glc), along with some (catechin)2-Mv3glc]; and the third group is their anthocyanin precursor, Mv3glc. (+info)
Changes in glycolytic enzyme activities in aging erythrocytes fractionated by counter-current distribution in aqueous polymer two-phase systems.
(14/131)
Human and rat erythrocytes were fractionated by counter-current distribution in charge-sensitive dextran/poly(ethylene glycol) two-phase systems. The specific activities of the key glycolytic enzymes (hexokinase, phosphofructokinase and pyruvate kinase) declined along the distribution profiles, although the relative positions of the activity profiles were reversed in the two species. These enzymes maintained their normal response to specific regulatory effectors in all cell fractions. No variations were observed for phosphoglycerate kinase and bisphosphoglycerate mutase activities. Some correlations between enzyme activities (pyruvate kinase/hexokinase, pyruvate kinase/phosphofructokinase, pyruvate kinase/pyruvate kinase plus phosphoglycerate kinase, pyruvate kinase/bisphosphoglycerate mutase and phosphoglycerate kinase/bisphosphoglycerate mutase ratios) were studied in whole erythrocyte populations as well as in cell fractions. These results strongly support the fractionation of human erythrocytes according to cell age, as occurs with rat erythrocytes. (+info)
Comparison of micellar electrokinetic capillary chromatography and high performance liquid chromatography on fingerprint of Cnidium monnieri.
(15/131)
In our studies, micellar electrokinetic capillary chromatography (MEKC) was employed in fingerprint analysis of Cnidium monnieri for the first time. Average chromatography of 10 batches Cnidium monnieri from Jiangsu province, China, which have long been considered as the original and genuine herbal medicine, was first established as the characteristic fingerprint. Within 25 min the major effective components were separated by 18 mM borate, 12 mM phosphate and 50 mM SDS (pH 9.2) containing 20% methanol. The relative standard deviations of migration times and peak areas were less than 5%. As a new approach of fingerprint, MKCE was compared to the conventional approach-HPLC in our experiments. The fingerprint developed by HPLC comprised 8 peaks that were collected within 40 min. Relative standard deviation (RSD) values of retention times of corresponding peaks in HPLC analysis were very small (maximum 3% and average 0.9%). In conclusion, each two methods had its advantages and disadvantages. Furthermore, besides HPLC, MEKC as a feasible method, could be used in the development of fingerprint of Cnidium monnieri. (+info)
Counter-current chromatography based analysis of synergy in an anti-tuberculosis ethnobotanical.
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The crude extract of an Alaskan ethnobotanical plant, Oplopanax horridus, was subjected to counter-current chromatography (CCC), and the selected active regions were evaluated for their synergistic effects with an in vitro model of anti-tubercular efficacy. CCC as a support-free high-resolution separation method was employed to preclude potential irreversible absorption to a solid stationary phase. The microplate Alamar blue assay and the isobole method were used to measure the biological activity and eliminate dose-response dependent errors, respectively. Using the combination of CCC, bioassay and isobole method, significant synergistic effects were observed. Among the entire polarity range, fractions with distribution constant between 0.44 and 0.81 showed the most synergistic enhancement with an increase in potency by 108% for the recombined fractions. (+info)