Enhancement of Sodium Caprate on Intestine Absorption and Antidiabetic Action of Berberine

Berberine, a plant alkaloid used in traditional Chinese medicine, has a wide spectrum of pharmacological actions, but the poor bioavailability limits its clinical use. The present aim was to observe the effects of sodium caprate on the intestinal absorption and antidiabetic action of berberine. The in situ, in vitro, and in vivo models were used to observe the effect of sodium caprate on the intestinal absorption of berberine. Intestinal mucosa morphology was measured to evaluate the toxic effect of sodium caprate. Diabetic model was used to evaluate antidiabetic effect of berberine coadministrated with sodium caprate. The results showed that the absorption of berberine in the small intestine was poor and that sodium caprate could significantly improve the poor absorption of berberine in the small intestine. Sodium caprate stimulated mucosal-to-serosal transport of berberine; the enhancement ratios were 2.08, 1.49, and 3.49 in the duodenum, jejunum, and ileum, respectively. After coadministration, the area under the plasma concentration–time curve of berberine was increased 28% than that in the absence of sodium caprate. Furthermore, both berberine and coadministration with sodium caprate orally could significantly decrease fasting blood glucose and improve glucose tolerance in diabetic rats (P?P?相似文献   

Composition and rheological properties of beta-Lactoglobulin/pectin coacervates: effects of salt concentration and initial protein/polysaccharide ratio

The composition and rheological properties of beta-lactoglobulin/pectin coacervates have shown significant correlations with sodium chloride concentration (C(NaCl)) and initial protein/polysaccharide ratio (r). An increase of C(NaCl) from 0.01 to 0.21 M at r = 5:1 leads to the increase in both beta-lactoglobulin and pectin contents in the coacervates, which can be explained in terms of salt-enhanced effect at lower salt concentrations. Further increase of C(NaCl) from 0.21 to 0.41 M decreases the proportions of these two biopolymers in the coacervates, exhibiting salt-reduced effect at higher salt concentrations. Moreover, the stronger self-aggregation of beta-lactoglobulin with increasing salt concentration gives rise to a decreasing actual protein/polysaccharide ratio in the coacervates at 0.01-0.21 M C(NaCl) and r = 5:1. An increase of r from 5:1 to 40:1 often increases the actual amount of pectin chains in beta-lactoglobulin/pectin coacervates, but it exhibits a maximum in beta-lactoglobulin content at r = 20:1. A much higher storage modulus (G') than loss modulus (G' ') for all beta-lactoglobulin/pectin coacervates suggests the formation of highly interconnected gel-like structure. The values of G' increase as C(NaCl) increases from 0.01 to 0.21 M, whereas a further increase of C(NaCl) from 0.21 to 0.41 M causes G' values to decrease to much lower values. These results further disclose the salt-enhanced effect and the salt-reduced effect at low and high salt concentrations, respectively. On the other hand, increasing r from 5:1 to 40:1 favors the formation of stronger gel-like beta-lactoglobulin/pectin coacervates, which mainly originates from the higher actual amount of pectin chains in beta-lactoglobulin/pectin coacervates at higher r values.     

Time-dependent polymerization of beta-lactoglobulin through disulphide bonds at the oil-water interface in emulsions

Time-dependent intermolecular sulphydryl-disulphide interchange involving beta-lactoglobulin adsorbed at the oil-water interface in n-tetradecane-in-water emulsions (10 wt% oil, 0.5 wt% protein, pH 7.0) has been investigated using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). While only monomers are detected in the adsorbed protein immediately after emulsion formation with pure beta-lactoglobulin, on storing the emulsion the amount of polymerized beta-lactoglobulin and the sizes of the oligomers are found to increase with time. There is no polymerization of adsorbed protein in emulsions made with pure alpha-lactalbumin after 72 h, or in emulsions made with beta-lactoglobulin in the presence of a reagent (N-ethylmaleimide) for modifying sulphydryl groups. Analysis by two-dimensional SDS-PAGE of adsorbed protein from aged emulsions made with a mixture of alpha-lactalbumin + beta-lactoglobulin shows some linking by disulphide bonds between alpha-lactalbumin and beta-lactoglobulin at the interface. Taken together with earlier time-dependent surface viscosity measurements, the results indicate the important role of free sulphydryl groups in the development of the high surface viscoelasticity of adsorbed globular proteins at the oil-water interface.     

Relationships between conformation of beta-lactoglobulin in solution and gel states as revealed by attenuated total reflection Fourier transform infrared spectroscopy

Attenuated total reflection Fourier transform infrared spectroscopy (ATR FT-IR) has been used to compare the structure of beta-lactoglobulin, the major component of whey proteins, in solution and in its functional gel state. To induce variation in the conformation of beta-lactoglobulin under a set of gelling conditions, the effect of heating temperature, pH, and high pressure homogenization on the conformation sensitive amide I band in the infrared spectra of both solutions and gels has been investigated. The results showed that gelification process has a pronounced effect upon beta-lactoglobulin secondary structure, leading to the formation of intermolecular hydrogen-bonding beta-sheet structure as evidenced by the appearance of a strong band at 1614 cm(-1) at the expense of other regular structures. These results confirm that this structure may be essential for the formation of a gel network as it was previously shown for other globular proteins. However, this study reveals, for the first time, that there is a close relationship between conformation of beta-lactoglobulin in solution and its capacity to form a gel. Indeed, it is shown that conditions which promote predominance of intermolecular beta-sheet in solution such as pH 4, prevent the formation of gel in conditions used by increasing thermal stability of beta-lactoglobulin. On the basis of these findings, it is suggested that by controlling the extent of intermolecular beta-structure of the protein in solution, it is possible to modify the ability of protein to form a gel and as a consequence to control the properties of gels.     

Influence of preadsorbed milk proteins on adhesion of Listeria monocytogenes to hydrophobic and hydrophilic silica surfaces.

The adsorption of beta-lactoglobulin, bovine serum albumin, alpha-lactalbumin, and beta-casein for 8 h and beta-lactoglobulin and bovine serum albumin for 1 h at silanized silica surfaces of low and high hydrophobicity, followed by incubation in buffer and contact with Listeria monocytogenes, resulted in different numbers of cells adhered per unit of surface area. Adhesion to both surfaces was greatest when beta-lactoglobulin was present and was lowest when bovine serum albumin was present. Preadsorption of alpha-lactalbumin and beta-casein showed an intermediate effect on cell adhesion. Adsorption of beta-lactoglobulin for 1 h resulted in a generally lower number of cells adhered compared with the 8-h adsorption time, while the opposite result was observed with respect to bovine serum albumin. The adhesion data were explainable in terms of the relative rates of arrival to the surface and postadsorptive conformational change among the proteins, in addition to the extent of surface coverage in each case.     

Quantification of glutamine in proteins and peptides using enzymatic hydrolysis and reverse-phase high-performance liquid chromatography

An enzymatic method for hydrolyzing bovine milk proteins was developed. Purified milk proteins (alpha-lactalbumin, beta-lactoglobulin, and beta-casein) were hydrolyzed in 0.1 M Hepes buffer (pH 7.5) containing pronase E, aminopeptidase M, and prolidase at 37 degrees C for 20 h. Free glutamine and other amino acids were derivatized with phenylisothiocyanate and separated using a C18 Pico-Tag column. Amino acids were eluted from the column with an aqueous sodium acetate-acetonitrile gradient with detection at 254 nm. Glutamine recoveries from hydrolyzed alpha-lactalbumin, beta-lactoglobulin, and beta-casein were 78 +/- 4, 98 +/- 3, and 101 +/- 3% of the theoretical values, respectively. The recoveries of most amino acids were comparable with those obtained using acid hydrolysis, except for the recoveries of proline and acidic amino acids. These peptide bonds appeared to be resistant to enzymatic hydrolysis and also to inhibit the hydrolysis of adjacent amino acids. Free glutamine was found to be very stable (97% recovery) under the enzymatic hydrolysis conditions.     

Anaerobic digestion of long-chain fatty acids in UASB and expanded granular sludge bed reactors

Solutions of sodium caprate and sodium laurate were digested in upflow anaerobic sludge bed (UASB) reactors inoculated with granular sludge and in expanded granular sludge bed (EGSB) reactors. UASB reactors are unsuitable if lipids contribute 50% or more to the COD of waste water: the gas production rate required to obtain sufficient mixing and contact cannot be achieved. At lipid loading rates exceeding 2–3 kg COD m⁻³ day⁻¹, total sludge wash-out occurred. At lower loading rates the system was unreliable, due to unpredictable sludge flotation. EGSB reactors do fulfil the requirements of mixing and contact. They accommodate space loading rates up to 30 kg COD m⁻³ day⁻¹ during digestion of caprate or laurate as sole substrate, at COD removal efficiencies of 83–91%, and can be operated at hydraulic residence times of 2 h without any problems. Augmentation of granular sludge in lab-scale EGSB reactors was demonstrated. The new granules had excellent settling properties. Floating layer formation, as well as mixing characteristics in full-scale EGSB reactors require further research.     

The interaction between Beta-lactoglobulin and sodium N-dodecyl sulphate.

1. The binding of sodium n-dodecyl sulphate to beta-lactoglobulin was studied in the pH range 3.5-7.0 by equilibrium dialysis, ultracentrifugation and microcalorimetry. 2. At low binding concentrations (less than 30 bound surfactants anions per protein molecule) the complexes formed aggregates in solution. 3. At higher binding concentrations aggregation does not occur at low ionic strength (0.01 mol/litre), but continues at high ionic strength (0.1 mol/litre). 4. At 25 degrees C the enthalpy of interaction of sodium n-dodecyl sulphate with beta-lactoglobulin can be interpreted as the sum of the enthalpies of formation of a complex with 2 bound surfactant anions, with an enthalpy change of -9.5 kJ-mol-1 of bound surfactant, and complexes containing at least 22 bound surfactant anions, with limiting enthalpies per bound surfactant anion of -12.4 kJ-mol-1 at pH 3.5 and -3.25 kJ-mol-1 at pH 5.5. 5. The binding of surfactant and the enthalpy of interaction at pH 3.5 ARE NOT SIGNIFICANTLY AFFECTED BY THE ADDITION Of 8 M-urea. 6. The data indicate that at low binding concentrations the interaction is of an ionic nature, and is accompanied by a conformational change in the protein.     

Characterization of pH-induced transitions of beta-lactoglobulin: ultrasonic, densimetric, and spectroscopic studies.

Depending on solution conditions, beta-lactoglobulin can exist in one of its six pH-dependent structural states. We have characterized the acid and basic-induced conformational transitions between these structural states over the pH range of pH 1 to pH 13. To this end, we have employed high-precision ultrasonic and densimetric measurements coupled with fluorescence and CD spectroscopic data. Our combined spectroscopic and volumetric results have revealed five pH-induced transitions of beta-lactoglobulin between pH 1 and pH 13. The first transition starts at pH 2 and is not completed even at pH 1, our lowest experimental pH. This transition is followed by the dimer-to-monomer transition of beta-lactoglobulin between pH 2.5 and pH 4. The dimer-to-monomer transition is accompanied by decreases in volume, v degrees (-0.008(+/-0.003) cm3 x g(-1)), and adiabatic compressibility, k degrees (S) (-(0.7(+/-0.4))x10(-6) cm3 x g(-1) x bar(-1)). We interpret the observed changes in volume and compressibility associated with the dimer-to-monomer transition of beta-lactoglobulin, in conjunction with X-ray crystallographic data, as suggesting a 7 % increase in protein hydration, with the hydration changes being localized in the area of contact between the two monomeric subunits. The so-called N-to-Q transition of beta-lactoglobulin occurs between pH 4.5 and pH 6 and is accompanied by increases in volume, v degrees (0.004(+/-0.003) cm3 x g(-1)), and compressibility, k degrees (S) ((0.7(+/-0.4))x10(-6) cm3 x g(-1) x bar(-1)). The Tanford transition of beta-lactoglobulin is centered at pH 7.5 and is accompanied by a decrease in volume, v degrees (-0.006(+/-0.003) cm3 x g(-1)), and an increase in compressibility, k degrees (S) ((1.5(+/-0.5))x10(-6) cm3 x g(-1) x bar(-1)). Based on these volumetric results, we propose that the Tanford transition is accompanied by a 5 to 10 % increase in the protein hydration and a loosening of the interior packing of beta-lactoglobulin as reflected in a 12 % increase in its intrinsic compressibility. Finally, above pH 9, the protein undergoes irreversible base-induced unfolding which is accompanied by decreases in v degrees (-0.014(+/-0.003) cm3 x g(-1)) and k degrees (S) (-(7.0(+/-0.5))x10(-6) cm3 x g(-1) x bar(-1)). Combining these results with our CD spectroscopic data, we propose that, in the base-induced unfolded state of beta-lactoglobulin, only 80 % of the surface area of the fully unfolded conformation is exposed to the solvent. Thus, in so far as solvent exposure is concerned, the base-induced unfolded states of beta-lactoglobulin retains some order, with 20 % of its amino acid residues remaining solvent inaccessible.     

A Novel Oral Preparation of Hydroxysafflor Yellow A Base on a Chitosan Complex: A Strategy to Enhance the Oral Bioavailability

Hydroxysafflor yellow A (HSYA), the main active pharmaceutical ingredient of the safflower plant (Carthamus tinctorius L.), is a hydrophilic drug with low oral bioavailability (BA). The objective of the present study was to improve the oral BA of HSYA by formulation design. The effect of several pharmaceutical excipients on enhancing BA, including Poloxamer 188 (P188), sodium caprate (SC), sodium deoxycholate, and β-cyclodextrin (β-CD), was investigated through animal models. Sodium caprate, with a relative BA of 284.2%, was able to improve the oral BA of HSYA. Furthermore, HSYA can bind with chitosan (CS) by Coulomb attraction and form a HSYA-CS complex. The preparation process was optimized, and the binding rate reached 99.4%. HSYA granules were prepared using a HSYA-CS complex and SC. The results of the pharmacokinetics showed that the relative BA of HSYA granules was 476%, much higher than HSYA/SC.KEY WORDS: absorption enhancer, chitosan, hydroxysafflor yellow A (HSYA), oral bioavailability     

Formation of soluble oligomers and amyloid fibrils with physical properties of the scrapie isoform of the prion protein from the C-terminal domain of recombinant murine prion protein mPrP-(121-231)

Prion diseases are fatal neurodegenerative disorders associated with conformational conversion of the cellular prion protein, PrP(C), into a misfolded, protease-resistant form, PrP(Sc). Here we show, for the first time, the oligomerization and fibrillization of the C-terminal domain of murine PrP, mPrP-(121-231), which lacks the entire unstructured N-terminal domain of the protein. In particular, the construct we used lacks amino acid residues 106-120 from the so-called amyloidogenic core of PrP (residues 106-126). Amyloid formation was accompanied by acquisition of resistance to proteinase K digestion. Aggregation of mPrP-(121-231) was investigated using a combination of biophysical and biochemical techniques at pH 4.0, 5.5, and 7.0 and at 37 and 65 degrees C. Under partially denaturing conditions (65 degrees C), aggregates of different morphologies ranging from soluble oligomers to mature amyloid fibrils of mPrP-(121-231) were formed. Transmission electron microscopy analysis showed that roughly spherical aggregates were readily formed when the protein was incubated at pH 5.5 and 65 degrees C for 1 h, whereas prolonged incubation led to the formation of mature amyloid fibrils. Samples incubated at 65 degrees C at pH 4.0 or 7.0 presented an initial mixture of oligomers and protofibrils or fibrils. Electrophoretic analysis of samples incubated at 65 degrees C revealed formation of sodium dodecyl sulfate-resistant oligomers (dimers, trimers, and tetramers) and higher molecular weight aggregates of mPrP-(121-231). These results demonstrate that formation of an amyloid form with physical properties of PrP(Sc) can be achieved in the absence of the flexible N-terminal domain and, in particular, of residues 106-120 of PrP and does not require other cellular factors or a PrP(Sc) template.     

Ascorbate interacts with sodium selenite to increase glutathione peroxidase activity in selenium-deficient chick duodena cultured in vitro

Duodena from Selenium (Se)/vitamin E-depleted 19 d chick embryos were cultured in vitro for 0-30 h. The addition of sodium selenite to the culture medium was associated with increased selenium-dependent glutathione peroxidase (SeGSHpx) activity after 24 h of incubation. In the absence of Se or in the presence of sodium ascorbate supplementation alone, SeGSHpx activity showed a gradual decline over the same time period. When ascorbate was added, along with sodium selenite, SeGSHpx activity was increased earlier and to a greater extent than in the presence of Se alone. These observations show that ascorbate can influence the metabolism of sodium selenite, resulting in increased SeGSHpx activity.     

Effect of sodium valproate on the secretion of proopiomelanocortin derived peptides from cultured rat anterior pituitary cells

We examined effects of sodium valproate, a gamma amino butyric acid (GABA)-transaminase inhibitor, on the secretion of immunoreactive (IR)-ACTH and IR-beta-endorphin/LPH from cultured rat anterior pituitary cells to determine whether sodium valproate has a direct action on the secretion of ACTH and its related peptides from the cultured rat anterior pituitary gland. During the 3 h incubation, the basal secretion of IR-ACTH and IR-beta-endorphin/LPH decreased to 50.8% and 58.3%, respectively, of the control concentration after adding 10(-7) M sodium valproate into the incubation media and to 67.7% and 69.3%, respectively, of the control levels with 10(-8) M sodium valproate. However, sodium valproate at a concentration of 10(-6) M or 10(-9) M did not affect the basal concentration of IR-ACTH and IR-beta-endorphin/LPH. Sodium valproate at a concentration of 10(-7) M significantly attenuated the stimulated release of IR-ACTH and IR-beta-endorphin/LPH by 10(-9) or 10(-10) M of ovine corticotrophin releasing factor. These results indicate that sodium valproate could directly effect rat anterior pituitary cells to suppress both basal and stimulated release of proopiomelanocortin derived peptides and this supports the hypothesis that sodium valproate has a direct effect at the pituitary corticotroph in reducing plasma ACTH.     

The effect of alpha-lactalbumin and beta-lactoglobulin hydrolysates on the metabolic activity of Escherichia coli JM103

Bovine milk proteins alpha-lactalbumin (alpha-la) and beta-lactoglobulin (beta-lg) were hydrolysed with seven different proteolytic enzymes, and the effect of various hydrolysates on a genetically modified luminous Escherichia coli JM103 was tested in vitro with a bioluminescence assay for bacterial growth and metabolism. Undigested proteins did not inhibit the activity of tested E. coli JM103 at a concentration as high as 0.1 g ml-1. At the same concentrations, alpha-la hydrolysed with pepsin or trypsin and beta-lg hydrolysed with alcalase, pepsin or trypsin, showed a lower metabolic activity during the first 8 h of growth. The activity of E. coli JM103 in the presence of 25 mg ml-1 alpha-la or beta-lg hydrolysed with pepsin and trypsin was only 21% of the control after incubation for 6 h. The preliminary results indicated that ultrafiltration through 10 kDa and 1 kDa molecular mass cut-off membranes may be used to enrich bacteriostatic properties.     

Fibrillar beta-lactoglobulin gels: Part 1. Fibril formation and structure

As a prelude to experimental and theoretical work on the mechanical properties of fibrillar beta-lactoglobulin gels, this paper reports the structural characterization of beta-lactoglobulin fibrils by electron and atomic force microscopy (AFM), infrared and Raman spectroscopy, and powder X-ray diffraction. Aggregates formed by incubation of beta-lactoglobulin in various alcohol-water mixtures at pH 2, and in water-trifluoroethanol (TFE) at pH 7, were found to be wormlike (approximately 7 nm in width and <500 nm in length), with a "string-of-beads" appearance. Longer (approximately 7 nm in width, and >1 microm in length), smoother, and seemingly stiffer fibrils formed on heating aqueous beta-lactoglobulin solutions at pH 2 and low ionic strength, although there was little evidence for the higher-order structures common in most amyloid-forming systems. Time-lapse AFM also revealed differences in the formation of these two fibril types: thermally induced aggregation occurring more cooperatively, in keeping with a nucleation and growth process. Only short stiff-rods (<20 nm in length) formed on heating beta-lactoglobulin at pH 7, and only complex three-dimensional "amorphous"aggregates in alcohols other than TFE at this pH. Studies of all of the pH 2 fibrils from beta-lactoglobulin, by Raman and infrared spectroscopy confirmed beta-sheet as mediating the aggregation process. Interestingly, however, some evidence for de novo helix formation for the solvent-induced systems was obtained, although it remains to be seen whether this is actually incorporated into the fibril-structure. In contrast to other amyloid systems, X-ray powder diffraction provided no evidence for extensive repeating "crystalline" structures for any of the pH 2 beta-lactoglobulin fibrils. In relation to amyloid, the lactoglobulin fibrils bear more resemblance to protofilaments than to higher-order fibril structures, these latter appearing more convincingly for thermally induced insulin fibrils (pH 2) also included in the AFM study.     

Isolation and complete primary sequence of a new ovine wild-type beta-lactoglobulin C

A new wild type of beta-lactoglobulin has been identified in the milk of sheep. It has been designated as ovine beta-lactoglobulin C. Its primary structure has been determined by direct protein microsequencing of intact protein and RP-HPLC-derived tryptic peptides. The new beta-lactoglobulin C is a subtype of ovine beta-lactoglobulin A with a single exchange Arg-Gln at position 148. This exchange may influence polymerisation of beta-lactoglobulin since in the crystal structure of orthorhombic bovine beta-lactoglobulin, residues 145-150 constitute a short beta-sheet region involved in dimer formation by pairing of dyad-related strands.     

Pressure denaturation of beta-lactoglobulin. Different stabilities of isoforms A and B, and an investigation of the Tanford transition.

Beta-lactoglobulin, the main whey protein in bovine milk, exists in several isoforms of which the most abundant are isoforms A and B. We have previously reported the denaturation of beta-lactoglobulin A by hydrostatic pressure [Valente-Mesquita, V.L., Botelho, M.M. & Ferreira, S.T. (1998) Biophys. J. 75, 471-476]. Here, we compare the pressure stabilities of isoforms A and B. These isoforms differ by two amino-acid substitutions: Asp64 and Val118 in isoform A are replaced by glycine and alanine, respectively, in isoform B. Replacement of the buried Val118 residue by the smaller alanine side-chain is not accompanied by significant structural rearrangements of the neighbouring polypeptide chain and creates a cavity in the core of beta-lactoglobulin. Pressure denaturation experiments revealed different stabilities of the two isoforms. Standard volume changes (DeltaVunf) of - 49 +/- 8 mL.mol-1 and -75 +/- 3 mL.mol-1, and unfolding free energy changes (DeltaGunf) of 8.5 +/- 1.3 kJ.mol-1 and 11.3 +/- 0.4 kJ.mol-1 were obtained for isoforms A and B, respectively. The volume occupied by the two methyl groups of Val118 removed in the V118A substitution is approximately 40 A3 per monomer of beta-lactoglobulin, in excellent agreement with the experimentally measured difference in DeltaVunf for the two isoforms (DeltaDeltaVunf = 26 mL.mol-1, corresponding to approximately 43 A3 per monomer). Thus, the existence of a core cavity in beta-lactoglobulin B may explain its enhanced pressure sensitivity relative to beta-lactoglobulin A. beta-Lactoglobulin undergoes a reversible pH-induced conformational change around pH 7, known as the Tanford transition. We have compared the pressure denaturation of beta-lactoglobulin A at pH 7 and 8. Unfolding free energy changes of 8.5 +/- 1.3 and 8.3 +/- 0.3 kJ.mol-1 were obtained at pH 7 and 8, respectively, showing that the thermodynamic stability of beta-lactoglobulin is identical at these pH values. Interestingly, DeltaVunf was dependent on pH, and varied from -49 +/- 8 mL.mol-1 to -68 +/- 2 mL.mol-1 at pH 7 and 8, respectively. The large increase in DeltaVunf at pH 8 relative to pH 7 appears to be associated with an overall expansion of the protein structure and could explain the increased pressure sensitivity of beta-lactoglobulin at alkaline pH.     

A highly thermostable and alkaline amylase from a Bacillus sp. PN5

A highly thermostable alkaline amylase producing Bacillus sp. PN5 was isolated from soil, which yielded 65.23 U mL(-1) of amylase in medium containing (%) 0.6 starch, 0.5 peptone and 0.3 yeast extract at 60 degrees C, pH 7.0 after 60 h of incubation. Maximum amylase activity was at pH 10.0 and 90 degrees C. The enzyme retained 80% activity after 1 h at pH 10.0. It exhibited 65% activity at 105 degrees C and had 100% stability in the temperature range between 80 and 100 degrees C for 1 h. In addition, there was 86.36% stability after 1-h incubation with sodium dodecylsulphate. These properties indicated possible use of this amylase in starch saccharification and detergent formulation.     

Response of human blood platelet membrane to sodium selenite.

It was demonstrated that incubation of blood platelets with sodium selenite (1-100 microM) resulted in a dose- and time-dependent loss of platelet thiols (both glutathione and protein -SH groups). The effects of sodium selenite on platelet membrane lipid fluidity by the EPR spin-labelling method was also investigated. We showed there were no alterations in membrane fluidity at the deeper regions (12-DOXYL-Ste) in lipid bilayer, a slight increase (approx. 7%, p < 0.03) of h +1/h0 for spin probe 5-DOXYL-Ste was monitored. The amount of Triton-insoluble protein fraction isolated from platelets after incubation (60 min) with selenite was significantly elevated (p < 0.006). It has been suggested that limited increase in lipid fluidity at the surface regions in the lipid bilayer of the platelet membrane in selenite-treated platelets may be the result of alteration in lipid-protein interactions caused by protein conformational changes.     

Formation of intracellular lumina in dispersed pig thyroid cells

Intracellular cavities characterized by the presence of microvilli have been identified in dispersed thyroid cells. These structures resembling follicular lumina were called intracellular lumina or ICL. Freshly dispersed cells did not contain ICL. At 37 degrees C, ICL formation was a rapid process. After 60 min of incubation, ICL were present in 15 to 20% of the cells; the number of ICL remained rather constant during 3 to 4 h of incubation. In the presence of thyrotropin, the number of ICL increased with time to reach a value ranging from 40 to 60 ICL per 100 cells after 4 h of incubation. ICL formation was also increased in the presence of dibutyryl cyclic AMP (2 mM). Vinblastine (30 microM), a microtubule-disrupting agent and monensin (30 microM), an ionophore inhibiting Golgi functions blocked the formation of ICL in control and thyrotropin-stimulated cells. Cycloheximide (0.5 mM) and puromycin (0.5 mM) did not inhibit ICL formation in either control or thyrotropin stimulated cells. The iodination capacity of ICL was studied by quantitative electron microscopic autoradiography after incubation of thyroid cells with 125 I-iodide for 2 to 60 min. Radioiodinated products appeared first in ICL. After 1 h of labeling autoradiographic grains were found mainly in ICL (60-70%) and over the cytoplasm. The labeling of ICL was heterogeneous; ICL contained either few or numerous overlapping grains. Whatever the labeling time, a high proportion of ICL (70-80%) were labeled. The labeling of ICL as well as the labeling over the cytoplasm was increased in the presence of thyrotropin and almost completely inhibited in the presence of an iodide trapping inhibitor: sodium perchlorate. Pulse-chase experiments revealed that thyrotropin stimulated the discharge of 125 I-labeled material from ICL.