Microemulsions Containing Medium-Chain Glycerides as Transdermal Delivery Systems for Hydrophilic and Hydrophobic Drugs

We evaluated the ability of microemulsions containing medium-chain glycerides as penetration enhancers to increase the transdermal delivery of lipophilic (progesterone) and hydrophilic (adenosine) model drugs as well as the effects of an increase in surfactant blend concentration on drug transdermal delivery. Microemulsions composed of polysorbate 80, medium-chain glycerides, and propylene glycol (1:1:1, w/w/w) as surfactant blend, myvacet oil as the oily phase, and water were developed. Two microemulsions containing different concentrations of surfactant blend but similar water/oil ratios were chosen; ME-lo contained a smaller concentration of surfactant than ME-hi (47:20:33 and 63:14:23 surfactant/oil/water, w/w/w). Although in vitro progesterone and adenosine release from ME-lo and ME-hi was similar, their transdermal delivery was differently affected. ME-lo significantly increased the flux of progesterone and adenosine delivered across porcine ear skin (4-fold or higher, p < 0.05) compared to progesterone solution in oil (0.05 +/- 0.01 mu g/cm(2)/h) or adenosine in water (no drug was detected in the receptor phase). The transdermal flux of adenosine, but not of progesterone, was further increased (2-fold) by ME-hi, suggesting that increases in surfactant concentration represent an interesting strategy to enhance transdermal delivery of hydrophilic, but not of lipophilic, compounds. The relative safety of the microemulsions was assessed in cultured fibroblasts. The cytotoxicity of ME-lo and ME-hi was significantly smaller than sodium lauryl sulfate (considered moderate-to-severe irritant) at same concentrations (up to 50 mu g/mL), but similar to propylene glycol (regarded as safe), suggesting the safety of these formulations.

Arterial heparan sulfate proteoglycans inhibit vascular smooth muscle cell proliferation and phenotype change in vitro and neointimal formation in vivo

Purpose: The aim of this study was to determine whether heparan sulfate proteoglycans (HSPGs) from the normal arterial wall inhibit neointimal formation after injury in vivo and smooth muscle cell (SMC) phenotype change and proliferation in vitro. Methods: Arterial HSPGs were extracted from rabbit aortae and separated by anion-exchange chromatography. The effect of HSPGs, applied in a periadventitial gel, on neointimal formation was assessed 14 days after balloon catheter injury of rabbit carotid arteries. Their effect on SMC phenotype and proliferation was measured by point-counting morphometry of the cytoplasmic volume fraction of myofilaments (Vvmyo) and H-3-thymidine incorporation in SMCs in culture. Results: Arterial HSPGs (680 mu g) reduced neointimal formation by 35% at 14 days after injury (P =.029), whereas 2000 mu g of the low-molecular-weight heparin Enoxaparin was ineffective. HSPGs at 34 mu g/mL maintained subconfluent primary cultured SMCs with the same high Vvmyo (52.1% +/- 13.8%) after 5 days in culture as did cells freshly isolated from the arterial wall (52.1% +/- 15.1%). In contrast, 100 mu g/mL Enoxaparin was ineffective in preventing phenotypic change over this time period (Vvmyo 38.9% +/- 14.6%, controls 35.9% +/- 12.8%). HSPGs also inhibited 3H-thymidine incorporation into primary cultured SMCs with an ID50 value of 0.4 mu g/mL compared with a value of 14 mu g/ml; for Enoxaparin (P

Cytotoxicity of pilosulin 1, a peptide from the venom of the jumper ant Myrmecia pilosula

The synthetic peptide pilosulin 1, corresponding to the largest defined allergenic polypeptide found in the venom of the jumper ant Myrmecia pilosula, inhibited the incorporation of [methyl-H-3]thymidine into proliferating Epstein-Barr transformed (EBV) B-cells. The LD50 was four-fold lower in concentration than melittin, a cytotoxic peptide found in honey bee venom. Loss of cell viability was assessed by flow cytometry by measuring the proportion of cells that fluoresced in the presence of the fluorescent dye 7-aminoactinomycin D. Examination of proliferating EBV B-cells indicated that the cells lost viability within a few minutes exposure to pilosulin 1. Partial peptides of pilosulin 1 were less efficient in causing loss of cell viability and the results suggest that the 22 N-terminal residues are critical to the cytotoxic activity of pilosulin 1. Normal blood white cells were also labile to pilosulin I. T- and B-lymphocytes, monocytes and natural killer cells, however, were more labile than granulocytes. Analysis of pilosulin I using circular dichroism indicated that, in common with melittin and other Hymenoptera venom toxins, it had the potential to adopt an cc-helical secondary structure. (C) 1998 Elsevier Science B.V, All rights reserved.

Structure-activity studies of conantokins as human N-methyl-D-aspartate receptor modulators

The activities of conantokin-G (con-G), conantokin-T (con-T), and several novel analogues have been studied using polyamine enhancement of [H-3]MK-801 binding to human glutamate-N-methyl-D-aspartate (NMDA) receptors, and their structures have been examined using CD and H-1 NMR spectroscopy. The potencies of con-G[A7], con-G, and con-T as noncompetitive inhibitors of spermine-enhanced [H-3]MK-801 binding to NMDA receptor obtained from human brain tissue are similar to those obtained using rat brain tissue. The secondary structure and activity of con-G are found to be highly sensitive to amino acid substitution and modification. NMR chemical shift data indicate that con-G, con-G[D8,D17], and con-G[A7] have similar conformations in the presence of Ca2+. This consists of a helix for residues 2-16, which is kinked in the vicinity of Gla10. This is confirmed by 3D structure calculations on con-G[A7]. Restraining this helix in a linear form (i.e., con-G[A7,E10-K13]) results in a minor reduction in potency. Incorporation of a 7-10 salt-bridge replacement (con-G[K7-E10]) prevents helix formation in aqueous solution and produces a peptide with low potency. Peptides with the Leu5-Tyr5 substitution also have low potencies (con-G[Y5,A7] and con-G[Y5,K7]) indicating that Leu5 in con-G is important for full antagonist behavior. We have also shown that the Gla-Ala7 substitution increases potency, whereas the Gla-Lys7 substitution has no effect. Con-G and con-G[K7] both exhibit selectivity between NMDA subtypes from mid-frontal and superior temporal gyri, but not between sensorimotor and mid-frontal gyri. Asn8 and/or Asn17 appear to be important for the ability of con-G to function as an inhibitor of polyamine-stimulated [3H]MK-801 binding, but not in maintaining secondary structure. The presence of Ca2+ does not increase the potencies of con-G and con-T for NMDA receptors but does stabilize the helical structures of con-G, con-G[D8,D17], and, to a lesser extent, con-G[A7]. The NMR data support the existence of at least two independent Ca2+-chelating sites in con-G, one involving Gla7 and possibly Gla3 and the other likely to involve Gla10 and/or Gla14.

Observation of a cubic phase in mesoporous silicate films grown at the air/water interface

X-Ray diffraction is reported from mesoporous silicate films grown at the air/water interface. The films were studied both as powdered films, and oriented on silicon or mica sheets. At early stages of growth we observe Bragg diffraction from a highly ordered cubic phase, with both long and short d-spacing peaks. We have assigned this as a discontinuous micellar Pm3n phase in which the silica is partly ordered. Later films retain only the known hexagonal p6m peaks and have lost any order both at short d-spacings and the longer d-spacing Bragg peaks characteristic of the cubic structure. The silica framework is considerably expanded from that in bulk amorphous silica, average Si Si distances are some 30% greater. Incorporation of glycerol or polyethylene glycol preserves the earlier cubic structure. To be consistent with earlier, in situ, X-ray and neutron reflectivity data we infer that both structures are produced after a phase transition from a less-ordered him structure late in the induction phase. The structural relations between the film Pm3n and p6m phase(s) and the known bulk SBA-1 and MCM-41 phases are briefly discussed.

The use of crosslinking promoters in the gamma-radiolysis of poly(tetrafluoroethylene-co-perfluoromethylvinyl ether). I

Incorporation of 1 wt % of triallyl isocyanurate (TAIC) significantly enhanced the radiation crosslinking of the perfluoroelastomer, poly(tetrafluoroethylene-co-perfluoromethylvinyl ether) (TFE/PMVE). The dose for gelation was lowered by 70% with the presence of TAIC. The additive also improved the tensile properties of TFE/ PMVE both before and after crosslinking by irradiation. Higher radical yields were obtained with the presence of TAIC at 77 K, indicating the crosslinking promoter was acting as a radical trap. ESR studies showed that radiolysis of TAIC and subsequent photobleaching cleaved an allyl branch from the ring structure. Upon thermal annealing, an allyl radical on the TAIC molecule was observed. (C) 1999 John Wiley & Sons, Inc.

Glycine metabolism by plant roots and its occurrence in Australian plant communities

Soluble organic nitrogen, including protein and amino acids, was found to be a ubiquitous form of soil N in diverse Australian environments. Fine roots of species representative of these environments were found to be active in the metabolism of glycine. The ability to incorporate [N-15]glycine was widespread among plant species from subantarctic to tropical communities. In species from subantarctic herbfield, subtropical coral cay, subtropical rainforest and wet heathland, [N-15]glycine incorporation ranged from 26 to 45% of (NH4+)-N-15 incorporation and was 2- to 3-fold greater than (NO3-)-N-15 incorporation. Most semiarid mulga and tropical savanna woodland species incorporated [N-15]glycine and (NO3-)-N-15 in similar amounts, 18-26% of (NH4+)-N-15 incorporation. We conclude that the potential to utilise amino acids as N sources is of widespread occurrence in plant communities and is not restricted to those from low temperature regimes or where N mineralisation is limited. Seedlings of Hakea (Proteaceae) were shown to metabolise glycine, with a rapid transfer of N-15 from glycine to serine and other amino compounds. The ability to take up and metabolise glycine was unaffected by the presence of equimolar concentrations of NO3- and NH4+. Isonicotinic acid hydrazide (INH) did not inhibit the transfer of N-15-label from glycine to serine indicating that serine hydroxymethyltransferase was not active in glycine catabolism. In contrast aminooxyacetate (AOA) strongly inhibited transfer of N-15 from glycine to serine and labelling of other amino compounds, suggesting that glycine is metabolised in roots and cluster roots of Hakea via an aminotransferase.

Effect of temperature and a crosslinking promoter on the gamma-radiolysis of a perfluoroelastomer

Methods of promoting the radiation-induced cross linking of poly(tetrafluoro-ethylene-co-perfluoromethyl vinyl ether) (TFE/PMVE) have been investigated. Greater control of the crosslinking and chain-scission reactions was achieved by varying the radiolysis temperature. This was attributed to temperature affecting the mobilities of reactive species such as polymeric free radicals. These reactive species are precursors to radiation-induced cross links and chain-ends. Analysis of the sol/gel behaviour, tensile properties and FTIR indicated that the optimum temperature for the radiation crosslinking of TFE/PMVE, at a dose of 150 kGy, was 263 K. This temperature was 10 K below the glass transition temperature. Incorporation of 1 wt% triallyl isocyanurate (TAIC) greatly amplified the radiation crosslinking of TFE/PMVE, The dose for gelation was decreased by 70%, and the additive imparted superior mechanical properties compared to the neat irradiated TFE/PMVE. Electron spin resonance (ESR) measurements showed higher radical yields at 77 K with the 1 wt% TAIC, indicating that the crosslinking promoter was acting as a radical trap. (C) 1999 Society of Chemical Industry.

Water diffusion in hydroxyethyl methacrylate (HEMA)-based hydrogels formed by gamma-radiolysis

Polymer hydrogels based upon methacrylates are used extensively in the pharmaceutical industry, particularly as controlled release drug delivery systems. These materials are generally prepared by chemically initiated polymerization, but this can lead to the presence of unwanted initiator fragments in the polymer matrix. In the present work, initiation of polymerization by gamma-irradiation of hydroxyethyl methacrylate, with and without added crosslinkers, has been investigated, and the diffusion coefficients for water in the resulting polymers have been measured through mass uptake by the polymers. The diffusion of water in poly(hydroxyethyl methacrylate) at 310 K was found to be Fickian, with a diffusion coefficient of 1.96 +/- 0.1 x 10(11) m(2) s(-1) and an equilibrium water content of 58%, NMR imaging analyses confirmed the adherance to a Fickian model of the diffusion of water into polymer cylinders. The incorporation of small amounts (0.2-0.5 wt%) of added ethyleneglycol-dimethacrylate-based crosslinkers was found to have only a small effect on the diffusion coefficient and the equilibrium water content for the copolymers. (C) 1999 Society of Chemical Industry.

Tetraphyllidean metacestodes of teleosts of the Great Barrier Reef, and the use of in vitro cultivation to identify them

The tetraphyllidean metacestode diversity of 310 teleost fishes, including 87 species from 31 families, was examined from Heron Island, The Great Barrier Reef, Australia. Eleven metacestode 'types' were identified with the use of light microscopy. Host-specificity varied greatly among metacestode types. Incorporation of in vitro cultivation allowed generic identification for some types. Types 1 and 2 belong to Uncibilocularis Southwell, 1925, and have triloculate bothridia and one pair of Forked hooks with unequal prongs; Type 3 has quadriloculate bothridia. Hook development was insufficient to determine in which genus, Acanthobothrium van Beneden, 1849 or Calliobothrium van Beneden, 1850, this type may belong. Type 4 has unilocular bothridia with simple edges and belongs to Anthobothrium van Beneden, 1850. Type 5 has multiloculated bothridia which are invaginated within pouches. This type belongs to the Rhinebothriinae although its generic identity cannot be determined. The bothridia of Type 5 everted within 24 hours of in vitro cultivation and revealed the presence of two forms, one having 48 loculi per bothridium, the other 72 per bothridium. In vitro studies provide additional support for existing theories of onchobothriid scolex development.

Stochastic structure and individual-tree growth models

The majority of past and current individual-tree growth modelling methodologies have failed to characterise and incorporate structured stochastic components. Rather, they have relied on deterministic predictions or have added an unstructured random component to predictions. In particular, spatial stochastic structure has been neglected, despite being present in most applications of individual-tree growth models. Spatial stochastic structure (also called spatial dependence or spatial autocorrelation) eventuates when spatial influences such as competition and micro-site effects are not fully captured in models. Temporal stochastic structure (also called temporal dependence or temporal autocorrelation) eventuates when a sequence of measurements is taken on an individual-tree over time, and variables explaining temporal variation in these measurements are not included in the model. Nested stochastic structure eventuates when measurements are combined across sampling units and differences among the sampling units are not fully captured in the model. This review examines spatial, temporal, and nested stochastic structure and instances where each has been characterised in the forest biometry and statistical literature. Methodologies for incorporating stochastic structure in growth model estimation and prediction are described. Benefits from incorporation of stochastic structure include valid statistical inference, improved estimation efficiency, and more realistic and theoretically sound predictions. It is proposed in this review that individual-tree modelling methodologies need to characterise and include structured stochasticity. Possibilities for future research are discussed. (C) 2001 Elsevier Science B.V. All rights reserved.

Structuring real-time Object-Z specifications

This paper presents a means of structuring specifications in real-time Object-Z: an integration of Object-Z with the timed refinement calculus. Incremental modification of classes using inheritance and composition of classes to form multi-component systems are examined. Two approaches to the latter are considered: using Object-Z's notion of object instantiation and introducing a parallel composition operator similar to those found in process algebras. The parallel composition operator approach is both more concise and allows more general modelling of concurrency. Its incorporation into the existing semantics of real-time Object-Z is presented.

A coiled-coil region of an insect immune suppressor protein is involved in binding and uptake by hemocytes

Polydnaviruses are associated with certain parasitoid wasps and are introduced into the body cavity of the host caterpillar during oviposition. Some of the viral genes are expressed in host tissues and corresponding proteins are secreted into the hemocoel causing suppression of the host immune system. The Cotesia rubecula polydnavirus gene product, CrV1, effectively inactivates hemocytes by mediating cytoskeleton break-down. A precondition for the CrV1 function is the incorporation of the extracellular protein by hemocytes. Here, we show that a coiled-coil domain containing a putative leucine zipper is required for CrV1 function, since removal of this domain abolishes binding and uptake of the CrV1 protein by hemocytes. (C) 2002 Elsevier Science Ltd. All rights reserved.

Increased generation of dendritic cells from myeloid progenitors in autoimmune-prone nonobese diabetic mice

Aberrant dendritic cell (DC) development and function may contribute to autoimmune disease susceptibility. To address this hypothesis at the level of myeloid lineage-derived DC we compared the development of DC from bone marrow progenitors in vitro and DC populations in vivo in autoimmune diabetes-prone nonbese diabetic (NOD) mice, recombinant congenic nonbese diabetes-resistant (NOR) mice, and unrelated BALB/c and C57BL/6 (BL/6) mice. In GM-CSF/IL-4-supplemented bone marrow cultures, DC developed in significantly greater numbers from NOD than from NOR, BALB/c, and BL/6 mice. Likewise, DC developed in greater numbers from sorted (lineage(-)IL-7Ralpha(-)SCA-1(-)c-kit(+)) NOD myeloid progenitors in either GM-CSF/IL-4 or GM-CSF/stem cell factor (SCF)/TNF-alpha. [H-3]TdR incorporation indicated that the increased generation of NOD DC was due to higher levels of myeloid progenitor proliferation. Generation of DC with the early-acting hematopoietic growth factor, flt3 ligand, revealed that while the increased DC-generative capacity of myeloid-committed progenitors was restricted to NOD cells, early lineage-uncommitted progenitors from both NOD and NOR had increased DC-gencrative capacity relative to BALB/c and BL/6. Consistent with these findings, NOD and NOR mice had increased numbers of DC in blood and thymus and NOD had an increased proportion of the putative myeloid DC (CD11c(+)CD11b(+)) subset within spleen. These findings demonstrate that diabetes-prone NOD mice exhibit a myeloid lineage-specific increase in DC generative capacity relative to diabetes-resistant recombinant congenic NOR mice. We propose that an imbalance favoring development of DC from myeloid-committed progenitors predisposes to autoimmune disease in NOD mice.

Identification of cellular changes associated with increased production of human growth hormone in a recombinant Chinese hamster ovary cell line

A proteomics approach was used to identify the proteins potentially implicated in the cellular response concomitant with elevated production levels of human growth hormone in a recombinant Chinese hamster ovary (CHO) cell line following exposure to 0.5 mM butyrate and 80 muM zinc sulphate in the production media. This involved incorporation of two-dimensional (2-D) gel electrophoresis and protein identification by a combination of N-terminal sequencing, matrix-assisted laser desorption/ionisation-time of flight mass spectrometry, amino acid analysis and cross species database matching. From these identifications a CHO 2-D reference,map and annotated database have been established. Metabolic labelling and subsequent autoradiography showed the induction of a number of cellular proteins in response to the media additives butyrate and zinc sulphate. These were identified as GRP75, enolase and thioredoxin. The chaperone proteins GRP78, HSP90, GRP94 and HSP70 were not up-regulated under these conditions.