An enclosed-chamber labeling system for the safe <Superscript>14</Superscript>C-enrichment of phytochemicals in plant cell suspension cultures

Summary Various plant secondary products have been implicated in the promotion of good health or the prevention of disease in humans, but little is known about the way they are absorbed in the gut, or in which tissues they are deposited throughout the body. While these issues could be studied if the phytochemicals were isotopically labeled, generating labeled molecules often is problematic because many compounds of interest can be synthesized only in planta at present. In order to generale ¹⁴C-labeled phytochemicals of high radioactive enrichment, we developed an enclosed-chamber labeling system in which cell suspension cultures can be safely and efficiently grown when supplied with ¹⁴C-enriched precursors. The system is designed to hold culture flasks within a clear, polyacrylic compartment that is affixed to the top of a rotary shaker. The flow-through gas exchange nature of the system allows for O₂ replenishment and complete capture of respired ¹⁴CO₂ throughout the entire period of cell culture. Air is circulated internally with the aid of a small fan, and chamber air temperature is monitored continuously with an internal temperature probe and data logger. Production runs of 12–14 d with Vaccinium pahalae (ohelo berry) and Vitis vinifera (grape) suspension cultures, using [¹⁴C]sucrose as the carbon source, demonstrated a 20–23% efficiency of ¹⁴C incorporation into the flavonoid-rich fractions. Further studies with ohelo cell cultures showed that flavonoids were produced with either sucrose or glucose as the carbohydrate source, although flavonoid productivity (measured as anthocyanins) was higher with sucrose. This comprehensive chamber system should have broad applicability with numerous cell types and can be used to generate a wide array of labeled phytochemicals.     

Preliminary brain-targeting studies on intranasal mucoadhesive microemulsions of sumatriptan

The aim of this investigation was to prepare microemulsions containing sumatriptan (ST) and sumatriptan succinate (SS) to accomplish rapid delivery of drug to the brain in acute attacks of migraine and perform comparative in vivo evaluation in rats. Sumatriptan microemulsions (SME)/sumatriptan succinate microemulsions (SSME) were prepared using titration method and characterized for drug content, globule size and size distribution, and zeta potential. Biodistribution of SME, SSME, sumatriptan solution (SSS), and marketed product (SMP) in the brain and blood of Swiss albino rats following intranasal and intravenous (IV) administrations were examined using optimized technetium-labeled (^99mTc-labeled) ST formulations. The pharmacokinetic parameters, drug targeting efficiency (DTE), and direct drug transport (DTP) were derived. Gamma scintigraphy imaging of rat brain following IV and intranasal administrations were performed to ascertain the localization of drug. SME and SSME were transparent and stable with mean globule size 38±20 nm and zeta potential between −35 to −55 mV. Brain/blood uptake ratios at 0.5 hour following IV administration of SME and intranasal administrations of SME, SMME, and SSS were found to be 0.20, 0.50, 0.60, and 0.26, respectively, suggesting effective transport of drug following intranasal administration of microemulsions. Higher DTE and DTP for mucoadhesive microemulsions indicated more effective targeting following intranasal administration and best brain targeting of ST from mucoadhesive microemulsions. Rat brain scintigraphy endorsed higher uptake of ST into the brain. Studies conclusively demonstrated rapid and larger extent of transport of microemulsion of ST compared with microemulsion of SS, SMP, and SSS into the rat brain. Hence, intranasal delivery of ST microemulsion developed in this investigation can play a promising role in the treatment of acute attacks of migraine. Published: January 20, 2006     

Balancing charge in the complementarity-determining regions of humanized mAbs without affecting pI reduces non-specific binding and improves the pharmacokinetics

Lowering the isoelectric point (pI) through engineering the variable region or framework of an IgG can improve its exposure and half-life via a reduction in clearance mediated through non-specific interactions. As such, net charge is a potentially important property to consider in developing therapeutic IgG molecules having favorable pharmaceutical characteristics. Frequently, it may not be possible to shift the pI of monoclonal antibodies (mAbs) dramatically without the introduction of other liabilities such as increased off-target interactions or reduced on-target binding properties. In this report, we explored the influence of more subtle modifications of molecular charge on the in vivo properties of an IgG1 and IgG4 monoclonal antibody. Molecular surface modeling was used to direct residue substitutions in the complementarity-determining regions (CDRs) to disrupt positive charge patch regions, resulting in a reduction in net positive charge without affecting the overall pI of the mAbs. The effect of balancing the net positive charge on non-specific binding was more significant for the IgG4 versus the IgG1 molecule that we examined. This differential effect was connected to the degree of influence on cellular degradation in vitro and in vivo clearance, distribution and metabolism in mice. In the more extreme case of the IgG4, balancing the charge yielded an ∼7-fold improvement in peripheral exposure, as well as significantly reduced tissue catabolism and subsequent excretion of proteolyzed products in urine. Balancing charge on the IgG1 molecule had a more subtle influence on non-specific binding and yielded only a modest alteration in clearance, distribution and elimination. These results suggest that balancing CDR charge without affecting the pI can lead to improved mAb pharmacokinetics, the magnitude of which is likely dependent on the relative influence of charge imbalance and other factors affecting the molecule''s disposition.     

Expression of a costly, plastic secondary sexual trait is correlated with age and condition in a damselfly with two male morphs

Males of the damselfly Mnais costalis Selys (Odonata: Calopterygidae) are morphologically and behaviourally polymorphic, typically existing as clear-winged non-territorial ‘sneaks’ and orange-winged territorial ‘fighters’. The amount of orange pigment in the wing, as measured with a chromameter, varied between individuals, and decreased as the reproductive season progressed. Young individuals maintained in the laboratory on high or low nutrient diets differed in the amount of pigment that developed in the wing. Males in the high nutrient group developed darker wings faster than those in the low nutrient group. Young adults of both sexes and morphs were fed ¹⁴C-radiolabelled tryptophan or tyrosine (precursors of the pigments ommochrome and melanin, respectively). Ommochrome was restricted to the pseudopterostigma of the males of both morphs and was not present in females. The presence of tyrosine in the wing cells of orange males, but not of clear males, indicated that the orange pigment is at least partly constituted from melanin. These data show that at least some pigment levels must be maintained continuously in the wings of orange males, and that maintenance is costly as it is compromised at low nutrient levels.     

RISING WATER TEMPERATURES ALTER LIPID DYNAMICS AND REDUCE N‐3 ESSENTIAL FATTY ACID CONCENTRATIONS IN SCENEDESMUS OBLIQUUS (CHLOROPHYTA)1

The biosynthesis of nutritionally important polyunsaturated fatty acids (PUFAs) in phytoplankton is influenced by environmental temperature. We investigated the potential of climate warming to alter lipid dynamics of Scenedesmus obliquus (Turpin) Kütz. by comparing lipid and fatty acid (FA) profiles as well as FA metabolism (using [1‐¹⁴C] acetate) at 20°C and 28°C. We documented an overall decline (53%–37%) in the proportion of n‐3 PUFA (in particular, of α‐linolenic acid [ALA; 18:3n‐3]), and a concomitant increase in saturated fatty acids (SAFAs) in total lipids (TLs) at 28°C, consistent with enhanced incorporation of radioactivity from [1‐¹⁴C] acetate into total 16:0, 18:1, and decreased incorporation into 18:2 and 18:3 FA (from 36% to 22% of the total) at 28°C. Glycerophospholipids were also affected by warming; ALA and stearidonic acids (SDAs; 18:4n‐3) both decreased (by 13% and 15%, respectively) in phosphatidylcholine (PC) and (by 24% and 20%, respectively) in phosphatidylethanolamine (PE). The characteristic FA in phosphatidylglycerol (PG; 16:1n‐13t) increased (by 22%) at 28°C. The activities of desaturases, which add double bonds to FA moieties, comprised the major suite of reactions affected by the temperature increase in TL and polar lipid (PL) classes. Climate modelers predict an increase in the number of extreme heat days in summer at temperate latitudes, with parallel projected increases in water temperatures of shallow water bodies. Our results suggest that the overall decrease in the essential n‐3 FA ALA in S. obliquus at higher water temperatures may lower food quality for higher tropic levels, adding another climate‐warming stress.     

Reconstitution Of β-catenin Degradation In Xenopus Egg Extract

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has been used to study protein turnover in many cellular contexts, including the cell cycle and signal transduction pathways^1-3. Herein, a method is described for isolating Xenopus egg extract that has been optimized to promote the degradation of the critical Wnt pathway component, β-catenin. Two different methods are described to assess β-catenin protein degradation in Xenopus egg extract. One method is visually informative ([³⁵S]-radiolabeled proteins), while the other is more readily scaled for high-throughput assays (firefly luciferase-tagged fusion proteins). The techniques described can be used to, but are not limited to, assess β-catenin protein turnover and identify molecular components contributing to its turnover. Additionally, the ability to purify large volumes of homogenous Xenopus egg extract combined with the quantitative and facile readout of luciferase-tagged proteins allows this system to be easily adapted for high-throughput screening for modulators of β-catenin degradation.     

Influence of poly(ethylene glycol) grafting density and polymer length on liposomes: Relating plasma circulation lifetimes to protein binding

The incorporation of poly(ethylene glycol) (PEG)-conjugated lipids in lipid-based carriers substantially prolongs the circulation lifetime of liposomes. However, the mechanism(s) by which PEG-lipids achieve this have not been fully elucidated. It is believed that PEG-lipids mediate steric stabilization, ultimately reducing surface-surface interactions including the aggregation of liposomes and/or adsorption of plasma proteins. The purpose of the studies described here was to compare the effects of PEG-lipid incorporation in liposomes on protein binding, liposome-liposome aggregation and pharmacokinetics in mice. Cholesterol-free liposomes were chosen because of their increasing importance as liposomal delivery systems and their marked sensitivity to protein binding and aggregation. Specifically, liposomes containing various molecular weight PEG-lipids at a variety of molar proportions were analyzed for in vivo clearance, aggregation state (size exclusion chromatography, quasi-elastic light scattering, cryo-transmission and freeze fracture electron microscopy) as well as in vitro and in vivo protein binding. The results indicated that as little as 0.5 mol% of 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine (DSPE) modified with PEG having a mean molecular weight of 2000 (DSPE-PEG₂₀₀₀) substantially increased plasma circulation longevity of liposomes prepared of 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC). Optimal plasma circulation lifetimes could be achieved with 2 mol% DSPE-PEG₂₀₀₀. At this proportion of DSPE-PEG₂₀₀₀, the aggregation of DSPC-based liposomes was completely precluded. However, the total protein adsorption and the protein profile was not influenced by the level of DSPE-PEG₂₀₀₀ in the membrane. These studies suggest that PEG-lipids reduce the in vivo clearance of cholesterol-free liposomal formulations primarily by inhibition of surface interactions, particularly liposome-liposome aggregation.     

Synthesis of selectively radiolabeled hexasaccharides for the determination of enzymatic regioselectivity

Poly-N-acetyllactosamines provide backbone structures for functional modifications such as sialyl Lewis X. To understand how the biosynthesis of poly-N-acetyllactosamines is regulated, two branched oligosaccharides of the structure Gal1,4GlcNAc1, 6(Gal1,4GlcNAc1,2)-Man1,6Man-octyl 1 and 2 were synthesized in which one of the terminal galactose units was selectively radiolabeled. Hexasaccharides 1 and 2 were assembled from the chemically synthesized pentasaccharide precursors GlcNAc1,6(Gal1,4GlcNAc1,2)-Man1,6Man-octyl3 and Gal1,4GlcNAc1,6(GlcNAc1, 2) - Man1,6 Man-octyl 4 respectively, through treatment with UDP-1-[³H]-Gal and 1,4 galactosyltransferase. Compounds 1 and 2 were subsequently incubated with UDP-GlcNAc and the UDP-GlcNAc: Gal1-4Glc(NAc)1,3-N-acetylglucosaminyltransferase (i-GlcNAc transferase) resulting in a partial conversion to a mixture of heptasaccharides which were purified by HPLC. The branch selectivity of the addition of N-acetylglucosamine to compounds 1 and 2 was then characterized by endo--galactosidase digestion of the heptasaccharides, followed by isolation of the resultant pentasaccharides on C18 reverse-phase silica cartridges. Comparison of the amount of radiolabel to a control reaction lacking endo--galactosidase indicated the favored site of GlcNAc addition to be the lower 1,2-branch over the 1,6-branch by a 3:1 ratio.     

Balancing charge in the complementarity-determining regions of humanized mAbs without affecting pI reduces non-specific binding and improves the pharmacokinetics

Lowering the isoelectric point (pI) through engineering the variable region or framework of an IgG can improve its exposure and half-life via a reduction in clearance mediated through non-specific interactions. As such, net charge is a potentially important property to consider in developing therapeutic IgG molecules having favorable pharmaceutical characteristics. Frequently, it may not be possible to shift the pI of monoclonal antibodies (mAbs) dramatically without the introduction of other liabilities such as increased off-target interactions or reduced on-target binding properties. In this report, we explored the influence of more subtle modifications of molecular charge on the in vivo properties of an IgG1 and IgG4 monoclonal antibody. Molecular surface modeling was used to direct residue substitutions in the complementarity-determining regions (CDRs) to disrupt positive charge patch regions, resulting in a reduction in net positive charge without affecting the overall pI of the mAbs. The effect of balancing the net positive charge on non-specific binding was more significant for the IgG4 versus the IgG1 molecule that we examined. This differential effect was connected to the degree of influence on cellular degradation in vitro and in vivo clearance, distribution and metabolism in mice. In the more extreme case of the IgG4, balancing the charge yielded an ～7-fold improvement in peripheral exposure, as well as significantly reduced tissue catabolism and subsequent excretion of proteolyzed products in urine. Balancing charge on the IgG1 molecule had a more subtle influence on non-specific binding and yielded only a modest alteration in clearance, distribution and elimination. These results suggest that balancing CDR charge without affecting the pI can lead to improved mAb pharmacokinetics, the magnitude of which is likely dependent on the relative influence of charge imbalance and other factors affecting the molecule's disposition.     

The production of antigens by Plasmodium falciparum in vitro

La, Lb, R¹ and S-antigens in extracts of human and Aotus monkey blood infected with Plasmodium falciparum were inactivated by several proteolytic enzymes but were not affected by nucleases and selected carbohydrases. S-antigens also survived oxidation by periodate under conditions which inactivated a known carbohydrate-associated antigen. It was concluded that the L, R and S-antigens are proteins.Autoradiographic studies of extracts of parasitized red cells which had been maintained in vitro in the presence of radioactive amino acids, showed that various La, Lb and R-antigens were labelled but S-antigens were not. The incorporation of labelled amino acids into antigens derived from parasitized mature mammalian red cells was taken as evidence that these antigens were of parasite origin whcreas the unlabelled S-antigens might be of host origin.