Spherical composite particles of rice starch and microcrystalline cellulose: A new coprocessed excipient for direct compression

Composite particles of rice starch (RS) and microcrystalline cellulose were fabricated by spray-drying technique to be used as a directly compressible excipient. Two size fractions of microcry stalline cellulose, sieved (MCS) and jet milled (MCJ), having volumetric mean diameter (D₅₀) of 13.61 and 40.51 μm, respectively, were used to form composite particles with RS in various mixing ratios. The composite particles produced were evaluated for their powder and compression properties. Although an increase in the microcrystalline cellulose proportion imparted greater compressibility of the composite particles, the shape of the particles was typically less spherical with rougher surface resulting in a decrease in the degree of flowability. Compressibility of composite particles made from different size fractions of microcrystalline cellulose was not different; however, using MCJ, which had a particle size range close to the size of RS (D₅₀=13.57 μm), provided more spherical particles than using MCS. Spherical composite particles between RS and MCJ in the ratio of 7∶3 (RS-MCJ-73) were then evaluated for powder properties and compressibility in comparison with some marketed directly compressible diluents. Compressibility of RS-MCJ-73 was greater than commercial spray-dried RS (Eratab), coprocessed lactose and microcrystalline cellulose (Cellactose), and agglomerated lactose (Tablettose), but, as expected, lower than microcrystalline cellulose (Vivapur 101). Flowability index of RS-MCJ-73 appeared to be slightly lower than Eratab but higher than Vivapur 101, Cellactose, and Tablettose. Tablets of RS-MCJ-73 exhibited low friability and good self-disintegrating property. It was concluded that these developed composite particles could be introduced as a new coprocessed direct compression excipient.     

Effect of freezing and thawing rates on denaturation of proteins in aqueous solutions

The freeze denaturation of model proteins, LDH, ADH, and catalase, was investigated in absence of cryoprotectants using a microcryostage under well-controlled freezing and thawing rates. Most of the experimental data were obtained from a study using a dilute solution with an enzyme concentration of 0.025 g/l. The dependence of activity recovery of proteins on the freezing and thawing rates showed a reciprocal and independent effect, that is, slow freezing (at a freezing rate about 1 degrees C/min) and fast thawing (at a thawing rate >10 degrees C/min) produced higher activity recovery, whereas fast freezing with slow thawing resulted in more severe damage to proteins. With minimizing the freezing concentration and pH change of buffer solution by using a potassium phosphate buffer, this phenomenon could be ascribed to surface-induced denaturation during freezing and thawing process. Upon the fast freezing (e.g., when the freezing rate >20 degrees C/min), small ice crystals and a relatively large surface area of ice-liquid interface are formed, which increases the exposure of protein molecules to the ice-liquid interface and hence increases the damage to the proteins. During thawing, additional damage to proteins is caused by recrystallization process. Recrystallization exerts additional interfacial tension or shear on the entrapped proteins and hence causes additional damage to the latter. When buffer solutes participated during freezing, the activity recovery of proteins after freezing and thawing decreased due to the change of buffer solution pH during freezing. However, the patterns of the dependence on freezing and thawing rates of activity recovery did not change except for that at extreme low freezing rates (<0.5 degrees C/min). The results exhibited that the freezing damage of protein in aqueous solutions could be reduced by changing the buffer type and composition and by optimizing the freezing-thawing protocol.     

Biodiversity of aquatic gastropods in the Mont St-Michel basin (France) in relation to salinity and drying of habitats

Seventy-seven polder and marsh stations were sampled for aquatic and riparian gastropods in the terrestrial basin of Mont St-Michel from March 1994–March 1995 and during a drought in 1996. In addition, four ponds with riparian coppice (bocage) were investigated. Twenty-one stations, mostly in polders, lacked snails; high values of conductivity, and by implication salinity, appeared to make the habitats unfavourable. Only the euryhaline hydrobid Potamopyrgus antipodarum and the amphibious lymnaeid Lymnaea truncatula survived in areas of high conductivity. In associated lagoons, the maximum level of salinity tolerated by freshwater gastropods was 35. Of the 14 species of gastropods collected, Lymnaea peregra and Anisus leucostoma were the most abundant. Stations had 1–7 species and the average gastropod species richness was 3.32 (±1.67). The canals that were the most susceptible to drying had a community of desiccation-resistant species including L. truncatula, Aplexa hypnorum, A. leucostoma and L. peregra. It appears that gastropod assemblages in the terrestrial basin of Mont St-Michel have achieved their current diversity by surviving strong ecological constraints.     

Oligomeric state of membrane transport proteins analyzed with blue native electrophoresis and analytical ultracentrifugation

Blue native electrophoresis is used widely for the analysis of non-dissociated protein complexes with respect to composition, oligomeric state and molecular mass. However, the effects of detergent or dye binding on the mass and stability of the integral membrane proteins have not been studied. By comparison with analytical ultracentrifugation, we have evaluated whether the oligomeric state of membrane transport proteins is reflected reliably with blue native electrophoresis. For the analysis we have used two well-characterized transporters, that is, the major facilitator superfamily protein LacS and the phosphotransferase system EII(Mtl). For another member of the major facilitator superfamily, the xyloside transporter XylP from Lactobacillus pentosus, the complete analysis of the quaternary structure determined by analytical ultracentrifugation and freeze-fracture electron microscopy is presented.Our experiments show that during blue native electrophoresis the detergent bound to the proteins is replaced by the amphipathic Coomassie brilliant blue (CBB) dye. The mass of the bound CBB dye was quantified. Provided this additional mass of bound CBB dye is accounted for and care is taken in the choice and concentration of the detergent used, the mass of LacS, XylP and EII(Mtl) and four other membrane (transport) proteins could be deduced within 10 % error. Our data underscore the fact that the oligomeric state of many membrane transport proteins is dimeric.     

Short-term effects of biological and physical forces on aggregate formation in soils with different clay mineralogy

The mechanisms resulting in the binding of primary soil particles into stable aggregates vary with soil parent material, climate, vegetation, and management practices. In this study, we investigated short-term effects of: (i) nutrient addition (Hoagland's solution), (ii) organic carbon (OC) input (wheat residue), (iii) drying and wetting action, and (iv) root growth, with or without dry–wet cycles, on aggregate formation and stabilization in three soils differing in weathering status and clay mineralogy. These soils included a young, slightly weathered temperate soil dominated by 2:1 (illite and chlorite) clay minerals; a moderately weathered soil with mixed [2:1 (vermiculite) and 1:1 (kaolinite)] clay mineralogy and oxides; and a highly weathered tropical soil dominated by 1:1 (kaolinite) clay minerals and oxides. Air-dried soil was dry sieved through a 250 m sieve to break up all macroaggregates and 100 g-subsamples were brought to field capacity and incubated for 42 days. After 14 and 42 days, aggregate stability was measured on field moist and air-dried soil, to determine unstable and stable aggregation respectively. In control treatments (i.e., without nutrient or organic matter addition, without roots and at constant moisture), the formation of unstable and stable macroaggregates (> 250 m) increased in the order: 2:1 clay soil < mixed clay soil < 1:1 clay soil. After 42 days of incubation, nutrient addition significantly increased both unstable and stable macroaggregates in the 2:1 and 1:1 clay soils. In all soils, additional OC input increased both unstable and stable macroaggregate formation. The increase in macroaggregation with OC input was highest for the mixed clay soil and lowest for the 1:1 clay soil. In general, drying and wetting cycles had a positive effect on the formation of macroaggregates. Root growth caused a decrease in unstable macroaggregates in all soils. Larger amounts of macroaggregates were found in the mixed clay and oxides soil when plants were grown under 50% compared to 100% field capacity conditions. We concluded that soils dominated by variable charge clay minerals (1:1 clays and oxides) have higher potential to form stable aggregates when OC concentrations are low. With additional OC inputs, the greatest response in stable macroaggregate formation occurred in soils with mixed mineralogy, which is probably a result of different binding mechanisms occurring: i.e., electrostatic bindings between 2:1 clays, 1:1 clays and oxides (i.e. mineral-mineral bindings), in addition to OM functioning as a binding agent between 2:1 and 1:1 clays.     

Preparation of dry powder dispersions for non-viral gene delivery by freeze-drying and spray-drying

Background

Dry powder dispersion devices offer potential for delivering therapeutic macromolecules to the pulmonary epithelia. Previously, freeze‐drying (lyophilisation) has been the accepted method for preparing dried formulations of proteins and non‐viral gene vectors despite the respirability of such powders being inadequate without further processing. In this study we compare the utility of freeze‐drying and spray‐drying, a one‐step process for producing dry and respirable powders, as methods for preparing non‐viral respiratory gene delivery systems.

Methods

Lipid:polycation:pDNA (LPD) vectors comprising 1,2‐dioleoyl‐3‐trimethylammoniumpropane (DOTAP), protamine sulphate and pEGFP‐N1 in 3% lactose solution were either snap‐frozen and lyophilised or spray‐dried. Lyophilised powder was used as recovered or following coarse grinding. Structural integrity of dehydrated pDNA was assessed by agarose gel electrophoresis and powder particle size determined by laser diffraction. The apparent structure of the systems was visualised by scanning and transmission electron microscopy with the biological functionality quantified in vitro (A549 human lung epithelial cell line) by Green Fluorescent Protein (GFP) associated fluorescence.

Results

Lyophilisation produced large, irregularly shaped particles prior to (mean diameter ～21 µm) and following (mean diameter ～18 µm) coarse grinding. Spray‐drying produced uniformly shaped spherical particles (mean diameter ～4 µm). All dehydrated formulations mediated reporter gene expression in A549 cells with the spray‐dried formulation generally proving superior even when compared with freshly prepared LPD complexes. Biological functionality of the LPD dry powders was not adversely affected following 3 months storage.

Conclusions

Spray‐drying has utility for producing stable, efficient and potentially respirable non‐viral dry powder systems for respiratory gene delivery. Copyright © 2002 John Wiley & Sons, Ltd.

     

Pure F-actin networks are distorted and branched by steps in the critical-point drying method

Elucidation of the ultrastructural organization of actin networks is crucial for understanding the molecular mechanisms underlying actin-based motility. Results obtained from cytoskeletons and actin comets prepared by the critical-point procedure, followed by rotary shadowing, support recent models incorporating actin filament branching as a main feature of lamellipodia and pathogen propulsion. Since actin branches were not evident in earlier images obtained by negative staining, we explored how these differences arise. Accordingly, we have followed the structural fate of dense networks of pure actin filaments subjected to steps of the critical-point drying protocol. The filament networks have been visualized in parallel by both cryo-electron microscopy and negative staining. Our results demonstrate the selective creation of branches and other artificial structures in pure F-actin networks by the critical-point procedure and challenge the reliability of this method for preserving the detailed organization of actin assemblies that drive motility.     

Stability of beta-carotene in spray dried preparation of Rhodotorula glutinis mutant 32

AIMS: To obtain beta-carotene-rich dry cell preparation from mutant 32 of Rhodotorula glutinis and determination of its pigment stability. METHODS AND RESULTS: The mutant 32 of R. glutinis was grown in a 14 l stirred tank fermenter. Cell mass was concentrated 10-fold by cross-flow microfiltration and then spray dried. Butylated hydroxy toluene (BHT) and d-tocopherol were used as protecting agents. A two-level, three-variable, factorial optimization was performed to achieve moisture-free, non-viable and beta-carotene-rich feed additive. CONCLUSIONS: The beta-carotene and cell mass in stirred tank fermenter were found to be 54 +/- 5 mg l-1 and 12.8 +/- 2 g l-1, respectively. In the presence of BHT, 97 +/- 3% (w/w) beta-carotene was recovered for all the inlet temperatures studied. The best beta-carotene and yeast powder recoveries were obtained at 160 degrees C, 11.6% (w/v) cell mass concentration and 1 g l-1 BHT. The pigments inside dried yeast powder were stable in dark and cold condition for at least 10 weeks. The purified beta-carotene got almost totally denatured, under similar conditions of storage, within 76 h. SIGNIFICANCE AND IMPACT OF THE STUDY: Spray dried and stable preparation of beta-carotene-rich yeast, R. glutinis can provide alternative source of beta-carotene for use in animal nutrition.     

Determination of the triacylglycerol composition of coffee beans by reverse-phase high-performance liquid chromatography

Reverse-phase HPLC with refractive index and light scattering detectors in isocratic and gradient elution modes, respectively, was applied for the separation of the major triacylglycerols (TAG) in coffee lipids. Twelve TAG species could be identified and determined using a linear gradient of acetonitrile in dichloromethane: dichloroethane. The quantitative evaluation was based on the relative area percentages derived directly from a data-station. The procedure was applied to determine the TAG composition of three types of coffee beans harvested in two coffee producing areas in Brazil and dried by two commonly used procedures. No significant differences in the TAG compositions due to the type, origin and drying procedure were found.