Poly(fumaric-co-sebacic) microspheres as oral drug delivery systems

The current study focuses on the development of bioadhesive oral delivery systems based on bioerodible polyanhydrides. The polymers were studied and characterized using a novel tensiometer based on a very sensitive electrobalance. The system was designed to mimic in vivo interactions, thus all experiments were conducted with freshly excised tissue immersed in physiological saline at 37 degrees C. Poly(fumaric-co-sebacic) [P(FA:SA)] was found to be the most bioadhesive polymer from a series of different thermoplastic materials evaluated. Correlation with in vivo performance was investigated by determining gastrointestinal (GI) residence time of barium-loaded microspheres. Residence times of 24 to 36 h provided a strong indication that these microspheres were good candidates for bioadhesive drug delivery systems. To evaluate the effect of these materials on bioavailability, the anticoagulant drug, dicumarol, was encapsulated. Systemic blood levels demonstrated increased bioavailability for the encapsulated dicumarol formulation as compared with unencapsulated drug. (c) 1996 John Wiley & Sons, Inc.     

Gram-scale purification of eicosapentaenoic acid (EPA, 20:5n-3) from wetPhaeodactylum tricornutum UTEX 640 biomass

Eicosapentaenoic acid (EPA, 20:5n-3) was obtained from the microalgaPhaeodactylum tricornutum following a three-step process: fatty acid extraction by direct saponification of wet biomass, polyunsaturated fatty acid (PUFA) concentration by formation of urea inclusion compounds and EPA isolation by preparative HPLC. Direct saponification of wet biomass was carried out with KOH-ethanol (96% v:v) (1 h, 60 °C), extracting 91% of the EPA. PUFAs were concentrated by the urea method with an urea/fatty acid ratio of 4:1 at a crystallization temperature of 28 °C using methanol as the urea solvent. An EPA concentration ratio of 1.5 (55.2/36.3) and recovery of 79% were obtained. This PUFA concentrate was used to obtain 95.8% pure EPA by preparative HPLC, using a reverse-phase column (C₁₈, 4.7 cm i.d. × 30 cm) and methanol-water (1% AcH) 80:20 w/w as the mobile phase. Ninety-seven per cent of EPA loaded was recovered and 70% EPA present in theP. tricornutum biomass was recovered in a highly pure form by means of this three-step downstream processing. In each of the HPLC preparative runs, 635 mg PUFA concentrate were loaded, obtaining 326 mg of a highly concentrated EPA fraction (2.46 g d^–1). Finally, a preliminary cost statement has been calculated.     

Attenuation effects on the kerma rates in air after cesium depositions on grasslands

Since the reactor accident of Chernobyl, cesium depth profiles and nuclide-specific kerma rates in air have been determined for various grassland sites in south Bavaria and in Ukraine. The sites are described by soil characteristics, annual precipitation, distance from release point, mode of deposition, and activity per unit area. The effects of surface roughness and migration of cesium into the soil on the kerma rate in air over grasslands was determined by two methods. The kerma rates in air obtained by the evaluations of in situ gamma-ray spectrometry results and of measured activity distributions in the soil showed only negligible differences for the observation period of 6 years after deposition. For the sites in Ukraine the kerma rate in air per activity per unit area was found to be systematically 40% higher than in Bavaria. The results from Bavaria on the attenuation of the kerma rate and a data set, including experiences from the weapons test fallout, are analytically approximated as a function of time up to 25 years after deposition.     

Interaction between cells and elastin fibers: An ultrastructural and immunocytochemical study

Mesenchymal cells (fibroblasts, smooth muscle cells) and endothelial cells were shown to interact with elastin fibers. The strong adhesion of elastin fibers to these cells is mediated by a cell membrane complex with a major glycoprotein component of 120 kDa designated as elastonectin. This interaction was studied by transmission electron microscopy (TEM) and immunocytochemical techniques using antibodies raised against the elastin adhesive proteins. When fibroblasts and smooth muscle cells were cultured in presence of elastin fibers, TEM showed an adhesion mechanism that takes place over several sites along the plasma membrane of these cells. Endothelial cells showed a very close association with elastin, emitting “pseudopodia” that embody the fibers. TEM, indirect immunofluorescence, immunoperoxidase, and confocal microscopy showed the presence and localization of cell membrane components synthesized in large quantities when cells were incubated in presence of elastin. Cells without elastin fibers barely revealed the adhesive membrane complex. These results confirm and extend previous findings concerning the presence of an inducible cell membrane complex that mediates the adhesion of elastin fibers to these cell types. © 1994 Wiley-Liss, Inc.     

Expression of carbohydrate residues in plasma membrane glycoproteins during the differentiation of amphibian epidermal cells

Summary Expression of various sugar residues on the plasma membrane of frog (Rana perezi) epidermal cells at different stages of differentiation has been monitored with the use of a battery of HRP-conjugated lectins. In paraffin-embedded tissue, mannose residues (stained by Concanavalin A) were detected at the keratinocyte cell surface in all epidermal strata. However,Lens culinaris agglutinin (LCA), also specific for mannose, specifically stained the plasma membrane of cells from the stratum germinativum. Expression of N-acetyl-glucosamine (GlcNAc), labelled with wheat germ agglutinin (WGA), was maximum at the cell surface of basal cells and progressively decreased through the stratum spinosum. Galactose (Gal) and N-acetyl-galactosamine (GalNAc) residues, labelled withGriffonia simplicifolia I (GS I) andGlycine max (SBA) agglutinins, respectively, were expressed according to the degree of differentiation in amphibian epidermal cells. Sialic acid-containing glycoproteins, labelled withLimax flavus agglutinin (LFA), were found in the outermost plasma membrane of the replacement cell layer and stratum corneum. Glycoproteins responsible for the observed lectin-binding patterns have been identified by staining on nitrocellulose filters after electrophoresis of solubilized plasma membrane fractions and Western blotting. Changes at the level of glycosylation of plasma membrane glycoproteins as epidermal cells differentiate are discussed on the basis of a progressive addition of Gal residues. Integral membrane proteins have been solubilized with the non-denaturing detergent CHAPS and glycoproteins containing terminal Gal residues, that are expressed according to the degree of differentiation in frog epidermis, have been partially purified by affinity chromatography on a GS I-Sepharose 4 B column. The purified fraction was composed by four acidic glycoproteins with isoelectric points between 4.6 and 5.2 and, in SDS-gels gave five major protein bands with approximate molecular weights of 148, 140, 102, 60, and 52 kDa in SDS-gels. The 102 and 52 kDa bands correspond to the a and subunits of amphibian epidermal Na⁺,K⁺-ATPase as demonstrated by specific staining with a polyclonal antibody against the catalytic subunit of pig kidney proton pump and staining with lectins GS I, GS II, and WGA. Possible relationships between higher molecular weight proteins and the constituents of intramembranous particles from the outermost plasma membranes of the replacement cell layer and the stratum corneum are also discussed.Abbreviations BSA bovine serum albumin - CHAPS (3-[(cholamidopropyl) dimethyl-ammonio] 1-propanesulfonate) - Con A Canavalia ensiformis agglutinin - DTT dithiothreitol - Gal galactose - GalNAc N-acetyl-D-galactosamine - GlcNAc N-acetyl-D-glucosamine - GS I Griffonia simplicifolia agglutinin I - GS II Griffonia simplicifolia agglutinin II - HRP horseradish peroxidase - LFA Limax flavus agglutinin - LCA Lens culinaris agglutinin - NDPAGIF non-denaturing polyacrylamide gel isoelectric focusing - PAGE polyacrylamide gel electrophoresis - PAP peroxidase-antiperoxidase - PBS phosphate buffered saline - PMSF phenyl methyl sulphonyl fluoride - RCL replacement cell layer - SBA soybean agglutinin (Glycine max) - SB stratum basal - SDS sodium dodecyl sulphate - SG stratum granulosum - SS stratum spinosum - UEA I Ulex europaeus agglutinin I - WGA wheat germ (Triticum vulgaris) agglutinin     

Simultaneous high-biomass protein production and nutrient removal using Spirulina maxima in sea water supplemented with anaerobic effluents

Maximum protein accumulation (71%, w/w) and nutrient removal by a mutant strain of Spirulina maxima growing on sea water supplemented with anaerobically treated pig slurry was achieved at 30°C with constant illumination (60 to 70 Em^-2s^-1), using a flow rate of 14.5 cm s^-1 (20 rev. min^-1 of a paddle wheel). Total phosphates were decreased by 99% and all ammonia-N was removed under these conditions.The authors are with the Department of Environmental Biotechnology, Institute of Ecology, Aptd Postal 63, Xalapa, Ver., Mexico     

Effect of photoperiod and exogenous melatonin on correlates of estrus in the domestic rabbit

It was the purpose of this study to test whether inhibition of estrus in the rabbit by short photoperiod could be simulated by subcutaneous implants of the pineal hormone melatonin. By measuring two non-invasive and readily quantifiable correlates of estrus, emission of the so-called nipple-search pheromone and scent-marking behavior (chinning), it was found that:

1. In intact does kept in stimulatory long photoperiod, melatonin suppressed chinning but had no effect on emission of nipple-search pheromone.
2. In intact, melatonin-treated does which were returned from inhibitory short day to stimulatory long day, chinning remained suppressed, whereas pheromone emission increased as for the vehicle-treated, control does.
3. In ovariectomized does, estradiol administration stimulated both pheromone emission and chinning whereas melatonin failed to suppress these effects.

Thus, although the administration of melatonin had an inhibitory effect on chinning it failed to suppress emission of the nipple-search pheromone and therefore failed to fully simulate the effect of short photoperiod on these two correlates of estrus. Several explanations for this discrepancy are considered, including the possibility that melatonin is not the sole mediary of the photoperiodic response, and that at least in female rabbits, other pineal products may also play a significant role.     

Expression of mutations and protein release by yeast conditional autolytic mutants in batch and continuous cultures

Thermosensitive mutant strains of Saccharomyces cerevisiae that fail to generate an osmotically stable cell wall when grown at a non-permissive temperature release their cell contents upon expression of the mutation. Therefore, they may represent an alternative for the production of homologous or heterologous protein preparations. In order to analyse the expression of two of these mutations, lyt2 and slt2, we grew the corresponding strains under precisely defined conditions in batch and continuous fermentors. A switch in the temperature of batch cultures from 24° C to 37° C determined lysis of the cells with a significant release of intracellular enzymes. These include alkaline phosphatase and periplasmic proteins such as glucan-degrading enzymes, the pattern of cell lysis and protein release being maintained for about 6 h. One-stage continuous cultures of a lyt2 mutant were maintained for long periods at 37° C; a fraction of the population lysed and released the indicated proteins, but eventually a revertant of the lytic phenotype was selected. To avoid this, a two-stage continuous culture system was developed by connecting two fermentors in series, the effluent from the first one at 24°C being fed to the second one adjusted to 37° C. A steady state of cell lysis and protein liberation was reached in the second-stage fermentor without any evidence of selection of revertants. This system can be very useful for developing conditions for the use of yeast strains to produce protein preparations. Correspondence to: C. Nombela     

Xylanolytic anaerobic thermophiles from Icelandic hot-springs

Anaerobic enrichment cultures inoculated with neutral and alkaline (pH 7.0–9.0) sediment and biomat samples from hot-springs in Hveragerdi and Fluir, Iceland, were screened for growth on beech xylan from pH 8.0 to 10.0 at 68° C: no growth occured in cultures above pH 8.4. Five anaerobic xylanolytic bacteria were isolated from enrichment cultures at pH 8.4; all five microbes were Gram-positive rods with terminal spores, and produced CO₂, H₂, acetate, lactate and ethanol from xylan and xylose. One of the isolates, strain A2, grew from 50 to 75° C, with optimum growth near 68° C, and from pH 5.2 to 9.0 with an optimum between 6.8 and 7.4. Taxonomically, strain A2 was most similar to Clostridium thermohydrosulfuricum. At pH 7.0, the supernatant xylanases of strain A2 had a temperature range from 50 to 78° C with an optimum between 68 and 78° C. At 68° C, xylanase activity occurred from pH 4.9 to 9.1, with an optimum from pH 5.0 to 6.6. At pH 7.0 and 68° C, the K _m of the supernatant xylanases was 2.75 g xylan/l and the V _max was 2.65 × 10^–6 kat/l culture supernatant. When grown on xylose, xylanase production was as high as when grown on xylan. Correspondence to: B. K. Ahring