Characterization of the mechanical properties of polymeric chromatographic particles by micromanipulation.

Chromatographic particles should not possess only desirable surface chemical properties but also mechanical properties. The latter determine the deformation of the particles under hydrodynamic conditions of packed beds and further the pressure drop. Understanding the mechanical properties of chromatographic particles is essential to successful design and operation of such processing equipment. The mechanical properties of single chromatographic particles made of different formulations and with different surface modifications were characterized by a novel micromanipulation technique. The principle of this technique is to compress single particles between two parallel surfaces. The force being imposed on the particles and their deformation under compression are measured simultaneously. The particles range from 30 to 300 microm, depending on their formulation. 10-30 particles from each sample were compressed up to a particle deformation of 70% in order to get statistically representative data. Particles for hydrophobic interaction chromatography were proved more stable than resins for ion exchange or gel filtration. The difference is remarkable in dry state and is less in wet state. Water seems to "level out" the differences in mechanical stability of resins. The stability seems to be also dependent on the speed of water release out of the resins.

The effect of the amount of binder liquid on the granulation mechanisms and structure of microcrystalline cellulose granules prepared by high shear granulation.

The structure of granules changes during the high shear granulation process. The purpose of this research was to investigate the effect of the amount of binder liquid on the structure of the granules and the structural changes which occur during the granulation process, using microcrystalline cellulose (MCC) and water as the model system. The structure is the result of the granulation mechanism; therefore, conclusions can be drawn about the latter by studying the former. X-ray microtomography and scanning electron microscopy (SEM) were applied in order to visualise the densification process of granules, which were first freeze dried in order to preserve their structure. Variations in their porosity were quantified by applying image analysis to the tomography results. In order to link the granule mechanical properties to their structural differences, a micromanipulation technique was used to measure granule resistance to deformation. MCC granules granulated with 100% (w/w) water showed increased densification with time, as expected; detailed examination showed that densification is more pronounced in the core of the granule; whereas the outer part remained more porous. Increased densification reduces deformability, so that granules become more resistant to breakage. The lower deformability of the densified granules in the final stages of granulation might result in establishment of equilibrium between attrition and growth, without substantial gross breakage. On the other hand, when more water was used (125%, w/w), densification was hardly observed; the porosity of the granule core was still high even after prolonged granulation times. This may be explained by the fact that higher water content increases the ease of deformation of granules. This increased deformability led to significant granule breakage even during the final phases of the granulation process. Therefore, for these granules a final equilibrium between breakage and coalescence might be established. This also explains why more granules produced with 125% granulation liquid were composed of fragments of irregular shape. Our results establish the link between the granulation behaviour of MCC in the latter stages and the material structure of these granules, which is determined by their liquid content. The process conditions (amount of liquid) to be chosen depend largely on the final purpose for which the granular material is produced.

Effect of polymerization conditions on the network properties of dex-HEMA microspheres and macro-hydrogels.

Dextran-hydroxy-ethyl-methacrylate (dex-HEMA) hydrogels in the form of microspheres are an attractive system for the controlled delivery of protein drugs. In this work, the microspheres were prepared by a water-in-water emulsion polymerization process. The polymerization reaction was initiated by potassium peroxodisulfate (KPS) and catalyzed by N,N,N',N'-tetramethylethylenediamine (TEMED). The effect of the initiator concentration, reaction temperature and pH on the mechanical and network properties of the microspheres were investigated. The size and size distribution of the microspheres, equilibrium water content, and methacrylate conversion were also determined. The mechanical properties of single microspheres were measured by a micromanipulation technique and the rheological characteristics of the same material in the form of macroscopic hydrogel slabs were determined by a controlled stress rheometer. The results showed that the Young's moduli of the microspheres and of macroscopic slabs measured by these two methods were in good agreement. Higher KPS initiator concentrations resulted in a more rapid polymerization with a shorter gelation and lag time, and a higher Young's modulus of the gels. An increase in temperature also resulted in a more rapid polymerization with a shorter gelation and lag time. However, the Young's modulus of the gels decreased with an increase in polymerization temperature. The pH had no significant effect on the mechanical properties of the microspheres. This study demonstrates that the network properties of dex-HEMA hydrogels can be tailored by the polymerization conditions, which opens the possibility to modulate the release rate of entrapped compounds.

Reversible changes in protein phosphorylation during germinal vesicle breakdown and pronuclear formation in bovine oocytes in vitro.

This study examined the event of protein phosphorylation in bovine oocytes during germinal vesicle breakdown (GVBD) and formation of pronuclei following fertilisation in vitro. Immature oocytes were obtained from abattoir materials and cultured in vitro. The oocytes were labelled with [32P]orthophosphate at 3 h intervals from 0 to 12 h following maturation in culture or from 3 to 18 h following insemination. One-dimensional gel electrophoresis indicated that levels of protein phosphorylation are low prior to GVBD. However, the levels of protein phosphorylation at approximately 40 kDa, 27 kDa, 23 kDa and 18 kDa increased substantially following GVBD and then decreased gradually as maturation in culture progressed. In contrast, the levels of protein phosphorylation increased gradually in the oocytes following pronucleus formation. Further, two-dimensional gel electrophoresis indicated that the protein at approximately 18 kDa reversibly changed in the oocytes during maturation and fertilisation. These results indicate that the reversible changes of this phosphoprotein may be related to either cell cycle transition or pronucleus formation during maturation and fertilisation in bovine oocytes.

Effect of centrifugation on early embryonic development and parthenogenetic activation of bovine oocytes matured in vitro.

This study examined the fertilization, early developmental competence and capacity for parthenogenetic activation of bovine oocytes matured in vitro after centrifugation. Immature oocytes were cultured in tissue culture medium 199 supplemented with 10% fetal bovine serum and 75 mIU mL(-1) FSH + LH at 5% CO2 to facilitate maturation. After culture for 24 or 30 h, the metaphase-II stage oocytes were centrifuged at 3000, 5000, 7000 or 10000g for 5 min before in vitro fertilization or parthenogenetic activation. Frozen-thawed bull semen was used for in vitro fertilization. For parthenogenetic activation, the oocytes were exposed to 20 microM calcium ionophore A23187 for 5 min at room temperature. Fertilization rates were not different between control and treatment groups (87.7% v. 74.6%, 73.4%, 75.9% and 76.4% respectively). Also, there were no differences in early embryonic development between control and treatment groups (rates of blastocyst formation were 21.1% v 20.2%, 28.8%, 31.2% and 24.1% respectively). When the oocytes were centrifuged at various speeds alone, the activation rate of oocytes was significantly higher (P < 0.05) in the 10000g treatment group compared with control (10.8% v 0.0%). There were no differences in the activation rates of oocytes between control and treatment groups at speeds up to 7000g (70.9% v. 71.9%, 78.3% and 77.2% respectively) after centrifugation and stimulation with Ca(2+)-ionophore. However, the activation rate of oocytes was significantly higher (P < 0.05) in the 10000g treatment group compared with control (70.9% v. 83.1%). In addition, the percentage of activated oocytes with diploid formation was significantly higher in the oocytes after centrifugation at 10000g and stimulation with calcium ionophore A23187 than in the control (18.4% v 7.1%). These results indicate that centrifugation of oocytes matured in vitro has no detrimental effect on fertilization and subsequent early embryonic development. They also indicate that the oocytes might be parthenogenetically activated after centrifugation and that high-speed centrifugation may induce activation of some oocytes. The results suggest that the optimal speed for centrifugation of bovine oocytes might be < or = 7000g to enhance the visibility of nuclear elements for further micromanipulation.

Activation of bovine oocytes following intracytoplasmic sperm injection (ICSI).

In the human and the mouse, intracytoplasmic sperm injection (ICSI) apparently triggers normal fertilization and may result in offspring. In the bovine, injection of spermatozoa must be accompanied by artificial methods of oocyte activation in order to achieve normal fertilization events (e.g., pronuclear formation). In this study, different methods of oocyte activation were tested following ICSI of in vitro-matured bovine oocytes. Bovine oocytes were centrifuged to facilitate sperm injection, and spermatozoa were pretreated with 5 mM dithiothreitol (DTT) to promote decondensation. Sperm-injected or sham-injected oocytes were activated with 5 microM ionomycin (A23187). Three hours after activation, oocytes with second polar bodies were selected and treated with 1.9 mM 6-dimethylaminopurine (DMAP). The cleavage rate of sperm-injected oocytes treated with ionomycin and DMAP was higher than with ionomycin alone (62 vs 27%, P < or = 0.05). Blastocysts (2 of 41 cleaved) were obtained only from the sperm-injected, ionomycin + DMAP-treated oocytes. Upon examination 16 h after ICSI, pronuclear formation was observed in 33 of 47 (70%) DMAP-treated oocytes. Two pronuclei were present in 18 of 33 (55%), while 1 and 3 pronuclei were seen in 8 of 33 (24%) and 7 of 33 (21%) oocytes, respectively. In sham-injected oocytes, pronuclear formation was observed in 15 of 38 (39%) with 9 (60%) having 2 pronuclei. Asa single calcium stimulation was insufficient and DMAP treatment could result in triploidy, activation by multiple calcium stimulations was tested. Three calcium stimulations (5 microM ionomycin) were given at 30-min intervals following ICSI. Two pronuclei were found in 12 of 41 (29%) injected oocytes. Increasing the concentration of ionomycin from 5 to 50 microM resulted in a higher rate of activation (41 vs 26%). The rate of metaphase III arrest was lower while the rate of pronuclear formation and cleavage development was higher in sperm-injected than sham-injected oocytes, suggesting that spermatozoa contribute to the activation process. Further improvements in oocyte activation following ICSI in the bovine are necessary.

Self-medication among secondary school pupils in Hong Kong: a descriptive study.

A self-completion questionnaire was used to survey self-medication among secondary school pupils in Hong Kong. Data were collected from 4793 pupils aged 10 to 23 years (55.9% female and 44.1% male). Nearly three quarters (72.1%) had taken self-medication without consulting a medical practitioner and 51.8% of the sample had done so without the knowledge of older family members. The prevalence of self-medication increased with age. More than half the pupils (50.4%) indicated that trivial illness did not warrant a consultation with a doctor. Information relating to the sources of self-administered drugs, types of drugs used and sources of information about these drugs was collected. Medicine cabinets at home and pharmacy shops were the two most common places from which the pupils obtained their drugs. Though the prevalence of taking tranquillizers and sleeping tablets was found to be low, the probability of young people, especially boys, obtaining dangerous drugs from these places should not be overlooked. The medical, nursing and teaching professions should take a more active role in health education, as the sources from which the pupils obtained their drug knowledge, in descending order of frequency, were: family members, previous illness experience, pharmacy shops, doctor or nurse, television or radio, newspapers or magazines, friends and teachers.

Bronchodilator effect of sodium cromoglycate and its clinical implications.

The bronchodilator effect of sodium cromoglycate (SCG) solution was investigated. Twenty asthmatic children aged 6-15 years (mean 11.3) were examined and the action of SCG compared with that of salbutamol and placebo (water). SCG produced a significantly raised peak expiratory flow rate (PEFR) before exercise, which reached a maximum immediately after exercise. The bronchodilatation was sustained for up to four hours, when the PEFR was still significantly above the resting value. This effect was comparable in degree and duration with that of salbutamol. In contrast, placebo produced insignificant bronchodilatation before exercise but significant albeit short-lived bronchodilatation after exercise, which is the characteristic response of the asthmatic to a short period of exercise. This powerful bronchodilator action of SCG and its equally potent inhibitory action on exercise-induced bronchoconstriction was achieved by administering the solution via an efficient nebuliser. In order to achieve maximum clinical effect the SCG must, therefore, be given in this form.