Active Uptake Mechanism Research Articles

Use of Plasma and Brain Unbound Fractions to Assess the Extent of Brain Distribution of 34 Drugs: Comparison of Unbound Concentration Ratios to in Vivo P-Glycoprotein Efflux Ratios

The P-glycoprotein (P-gp)-deficient mouse model is used to assess the influence of P-gp-mediated efflux on the central nervous system (CNS) distribution of drugs. The steady-state unbound plasma/unbound brain concentration ratio ([plasma],(u)/[brain],(u)) is an alternative method for assessing CNS distribution of drugs independent of the mechanism(s) involved. The objective of this study was to compare the degree of CNS distributional impairment determined from the in vivo P-gp efflux ratio with that determined from the [plasma],(u)/[brain],(u) ratio. CNS distribution of 34 drugs, including opioids, triptans, protease inhibitors, antihistamines, and other clinically relevant drugs with either poor CNS distribution or blood-brain barrier efflux, was studied. Plasma and brain unbound fractions were determined by equilibrium dialysis. K(p,brain) and the P-gp efflux ratio were obtained from the literature or determined experimentally. The P-gp efflux ratio and the [plasma],(u)/[brain],(u) ratio were in concurrence (<3-fold difference) for 21 of the 34 drugs. However, the [plasma],(u)/[brain],(u) ratio exceeded the P-gp efflux ratio substantially (>4-fold) for 10 of the 34 drugs, suggesting that other, non-P-gp-mediated mechanism(s) may limit the CNS distribution of these drugs. The P-gp efflux ratio exceeded the [plasma],(u)/[brain],(u) ratio by more than 3-fold for three drugs, suggesting the presence of active uptake mechanism(s). These observations indicate that when mechanisms other than P-gp affect CNS distribution (non-P-gp-mediated efflux, poor passive permeability, cerebrospinal fluid bulk flow, metabolism, or active uptake), the P-gp efflux ratio may underestimate or overestimate CNS distributional impairment. The [plasma],(u)/[brain],(u) ratio provides a simple mechanism-independent alternative for assessing the CNS distribution of drugs.

The impact of hypoxia on in vivo glucose uptake in a hypoglycemic fish,Myoxocephalus scorpius

The mechanisms controlling carbohydrate utilization in teleost fish are poorly understood, particularly in the heart. Tissue glucose uptake and cardiovascular characteristics were measured in the short-horned sculpin, Myoxocephalus scorpius, a species exhibiting low blood glucose levels, during normoxia and hypoxia to assess the role of adenosine receptors in the control of glucose uptake and anaerobic metabolism. As expected, hypoxia exposure (300 min at 2 mg/l dissolved oxygen) resulted in a bradycardia and plasma lactate accumulation, but glucose uptake rates did not change in heart, brain, gill, spleen, and white muscle. Plasma glucose-to-intracellular glucose ratios indicated that glucose uptake was the rate-limiting step in glucose utilization. The majority of intracellular glucose was unphosphorylated, however, suggesting that hexokinase is also important in controlling the tissue glucose gradient. During hypoxia, the cholinergic blocker atropine resulted in tachycardia but did not significantly change tissue glucose uptake rates or heart and brain adenosine levels. In contrast, the combined treatment of atropine and an adenosine receptor blocker [8-(p-sulfophenyl)theophylline] during hypoxia increased heart glucose uptake to levels fivefold higher than normoxic fish, with no additive effects on cardiovascular parameters. Significant tissue lactate accumulation was observed in this group of fish, signifying that adenosine receptors may depress anaerobic metabolism, even though tissue adenosine accumulation was absent during hypoxia. White muscle accumulated glucose during normoxia, suggesting the presence of gluconeogenic pathways or active uptake mechanisms not previously described in this tissue.

Water Balance Components in Adults of Terrestrial Red Mite &lt;I&gt;Balaustium&lt;/I&gt; sp. (Acarina: Erythraeidae)

Balaustium spp. mites are noted for their quickness, red color, population bursts, and ubiquity. Water balance characteristics of adults were assessed to determine their primary source of water to gain insight into the biology and physiology of these poorly studied mites. Characteristics of this mite show that it is xeric adapted, featuring a 71% body water content, 39% dehydration tolerance limit, and a modest water loss rate of 2% h−1. This species is unable to absorb water vapor from atmospheric air which is unusual for mites. This is illustrated by net water losses at water vapor activities close to saturation, failure to maintain the uptake after first day passive gains, and no detectable salt accumulation around the base of the mouthparts in scanning electron micrographs. Thus, enhanced water conservation enables this mite to withstand desiccation, and their aggressive predatory life style has apparently not necessitated evolution of an active uptake mechanism for absorption of water vapor for this mite that obtains most of its water from feeding.

Carbon acquisition mechanisms by planktonic desmids and their link to ecological distribution

To test if different inorganic carbon (Ci) uptake mechanisms underlie the ecological distribution pattern of planktonic desmids, we performed pH-drift experiments with 12 strains, belonging to seven species, originating from lakes of different pH. Staurastrum brachiatum Ralfs and Staurodesmus cuspidatus (Ralfs) Teil. var. curvatus (W. West) Teil., species confined to acidic, soft water habitats, showed remarkably different behavior in the pH drift experiments: S. brachiatum appeared to use CO2 only, whereas Staurodesmus cuspidatus appeared to use HCO3– as well. Staurastrum chaetoceras (Schr.) Smith and Staurastrum planctonicum Teil, species well-known for their abundant occurrence in alkaline waters, were the most effective at using HCO3–. Other species, to be encountered in both slightly acidic and slightly alkaline waters, took an intermediate position. Experiments using specific inhibitors suggested that Cosmarium abbreviatum Rac. var. planctonicum W. &amp; G.S. West and S. brachiatum use CO2 by an active CO2 uptake mechanism, whereas S. chaetoceras and Staurodesmus cuspidatus showed an active HCO3– uptake pattern. Most likely, these active uptake mechanisms make use of H+-ATPase, as none of the desmids expressed significant carbonic anhydrase activity. A series of strains of Staurastrum planctonicum isolated from different habitats, all clustered in between the species using HCO3–, but no further differentiation was observed. Therefore, desmids cannot be simply characterized as exclusive CO2 users, and the ecological distribution pattern of a desmid species does not unequivocally link to a certain Ci uptake mechanism. Nevertheless, there does appear to be a general ecological link between a species' Ci uptake mechanism and its ecological distribution.Key words: pH drift, desmids, isolate variation, inorganic carbon acquisition.

Human intestinal and lung cell lines exposed to β-carotene show a large variation in intracellular levels of β-carotene and its metabolites

Although in vitro models are often used in β-carotene research, knowledge about the uptake and metabolism of β-carotene in cell lines is lacking. We measured by HPLC the intracellular levels of β-carotene and its metabolites in 9 human intestinal and lung cell lines after exposure to 1 μM β-carotene during 2, 6, 30, 54 h, and 3 weeks. In three colorectal carcinoma cell lines only low levels of β-carotene could be detected and an apparent linear increase in intracellular β-carotene was observed during the whole exposure period of 3 weeks. The remaining cell lines (an SV40 transformed colon cell line, a small intestinal carcinoma cell line and several lung cell lines) had medium or high intracellular β-carotene levels. In these cell lines intracellular β-carotene quickly increased during the first 54 h of exposure and after 3 weeks no further increase was observed, suggesting a stable level of β-carotene after 54 h. Estimated intracellular concentrations at steady-state levels varied between 2 and 5 μM (low) or 9 and 55 μM (medium/high). Our results seem to indicate that an active uptake mechanism of β-carotene exists in at least a subset of cell lines. Seven different β-carotene metabolites were detected in the various cell lines ( cis-carotene, retinol, three epoxy-carotenes, and two retinyl esters). Metabolite levels were the highest in cells with medium or high β-carotene levels. Each cell line appeared to have a distinct metabolite profile. No intestinal or lung specific pattern could be found, but two epoxy-carotene metabolites were not detectable in the colon cell lines.

Elemental Uptake in Relation to Root Characteristics of Tall Fescue ##

HiMag, an accession of tall fescue (Festuca arundinacea Schreb.), was selected for high magnesium (Mg) concentration in leaves to reduce grass tetany risk to ruminants. However, the mechanism for enhanced Mg uptake in HiMag leaves has not been determined. The objective was to investigate if increased Mg uptake in HiMag could be explained by differences in elemental distribution among plant parts, root characteristics, or organic acid concentrations compared to its parental cultivars, “Kentucky 31” (KY31) and “Missouri 96” (MO96). The study was conducted on a surface-irrigated calcareous Portneuf silt loam (coarse-silty, mixed, mesic, Durinodic Xeric Haplocalcid). Vegetation and soil cores of 7.6-cm diameter were sampled to a 45-cm soil depth in 15-cm increments. Mass and ash were determined for leaves, crowns, and roots. Leaf area, root length, root area, root length density, elemental concentration, and uptake [potassium (K), calcium (Ca), Mg, sodium (Na), and phosphorus (P)], and malate and citrate concentrations also were determined. Leaf Mg concentration was higher in HiMag than parental cultivars. HiMag generally did not differ in crown and root elemental concentrations from its parents. Risk of causing grass tetany, indicated by leaf K/(Ca + Mg), was lower in HiMag than KY31 and MO96 in both 1994 (P = 0.03) and 1995 (P = 0.01). Root length, area, and mass were not related to cation concentrations in the three tall fescue accessions, suggesting that HiMag may have an active uptake or transport mechanism for Mg. #University of Idaho Ag. Expt. Sta. No. 01709.

Labile zinc and zinc transporter ZnT4 in mast cell granules: role in regulation of caspase activation and NF-kappaB translocation.

The granules of mast cells and other inflammatory cells are known to be rich in zinc (Zn), a potent caspase inhibitor. The functions of granular Zn, its mechanism of uptake, and its relationship to caspase activation in apoptosis are unclear. The granules of a variety of mast cell types fluoresced intensely with the Zn-specific fluorophore Zinquin, and fluorescence was quenched by functional depletion of Zn using a membrane-permeable Zn chelator N, N, N', N'-tetrakis (2-pyridyl-methyl)ethylenediamine (TPEN). Zn levels were also depleted by various mast cell activators, including IgE/anti-IgE, and Zn was rapidly replenished during subsequent culture, suggesting an active uptake mechanism. In support of the latter, mast cells contained high levels of the vesicular Zn transporter ZnT(4), especially in the more apical granules. Immunofluorescence and immunogold labeling studies revealed significant pools of procaspase-3 and -4 in mast cell granules and their release during degranulation. Functional depletion of Zn by chelation with TPEN, but not by degranulation, resulted in greatly increased susceptibility of mast cells to toxin-induced caspase activation, as detected using a fluorogenic substrate assay. Release of caspases during degranulation was accompanied by a decreased susceptibility to toxins. Zn depletion by chelation, but not by degranulation, also resulted in nuclear translocation of the antiapoptotic, proinflammatory transcription factor NF-kappaB. These findings implicate a role for ZnT(4) in mast cell Zn homeostasis and suggest that granule pools of Zn may be distinct from those regulating activation of procaspase-3 and NF-kappaB.

Bicarbonate uptake by Southern Ocean phytoplankton

Marine phytoplankton have the potential to significantly buffer future increases in atmospheric carbon dioxide levels. However, in order for CO2 fertilization to have an effect on carbon sequestration to the deep ocean, the increase in dissolved CO2 must stimulate primary productivity; that is, marine phototrophs must be CO2 limited [Riebesell et al., 1993]. Estimation of the extent of bicarbonate (HCO3−) uptake in the oceans is therefore required to determine whether the anthropogenic carbon sources will enhance carbon flux to the deep ocean. Using short‐term 14CO2‐disequilibrium experiments during the Southern Ocean Iron Experiment (SOFeX), we show that HCO3− uptake by Southern Ocean phytoplankton is significant. Since the majority of dissolved inorganic carbon (DIC) in the ocean is in the form of bicarbonate, the biological pump may therefore be insensitive to anthropogenic CO2. Approximately half of the DIC uptake observed was attributable to direct HCO3− uptake, the other half being direct CO2 uptake mediated either by passive diffusion or active uptake mechanisms. The increase in growth rates and decrease in CO2 concentration associated with the iron fertilization did not trigger any noticeable changes in the mode of DIC acquisition, indicating that under most environmental conditions the carbon concentrating mechanism (CCM) is constitutive. A low‐CO2 treatment induced an increase in uptake of CO2, which we attributed to increased extracellular carbonic anhydrase activity, at the expense of direct HCO3− transport across the plasmalemma. Isotopic disequilibrium experimental results are consistent with Southern Ocean carbon stable isotope fractionation data from this and other studies. Although iron fertilization has been shown to significantly enhance phytoplankton growth and may potentially increase carbon flux to the deep ocean, an important source of the inorganic carbon taken up by phytoplankton in this study was HCO3−, whose concentration is negligibly affected by the anthropogenic rise in CO2. We conclude that biological productivity in this region of the world's ocean is unlikely to be directly regulated by natural or anthropogenic variations in atmospheric CO2 concentrations because of the presence of a constitutive CCM.

Studies on Microbial Heavy Metal Retention from Uranium Mine Drainage Water with Special Emphasis on Rare Earth Elements

Microbial heavy metal retention was studied using seepage water sampled from a former uranium mining site in Eastern Thuringia, Germany. The seepage water has a low pH and contains high concentrations of metals, including uranium, rare earth elements (REE), and other heavy metals. Microbial influence on sorption and/or active uptake of heavy metals was studied using REE patterns. Incubation of seepage water with the bacterium Escherichia coli caused sorption of heavy metals to biomass. Incubation with the fungus Schizophyllum commune, however, had a much more pronounced effect, including significant fractionation of REE, pointing to the possibility of a specific active uptake mechanism. Extraction factors and fractionation coefficients are given to show the capacity of the presented bioextraction for future applications.

Basolateral active uptake of nitrofurantoin in the CIT3 cell culture model of lactation.

Nitrofurantoin and other agents are actively transported into human and rodent milk. The purpose of this study was to determine whether nitrofurantoin active transport across mammary epithelia occurs basolaterally or apically, using the CIT3 cell culture model of lactation. The CIT3 model actively transports nitrofurantoin in the basolateral to apical direction. Basolateral to apical permeability [92.9 +/- 6.6 (microl/h)/cm(2)] was differentially decreased by unlabeled nitrofurantoin (250 microM) on the basolateral, apical, or both sides [49.5 +/- 1.8, 57.9 +/- 1.4, or 48.5 +/- 1.6 (microl/h)/cm(2), respectively]. Apical to basolateral permeability [27.6 +/- 1.8 (microl/h)/cm(2)] was increased in the presence of unlabeled nitrofurantoin (250 microM) on the basolateral, apical, or both sides [36.4 +/- 1.5, 39.9 +/- 0.7, 42.4 +/- 1.1 (microl/h)/cm(2), respectively]. These data indicate a basolateral active uptake mechanism for nitrofurantoin, which remains to be identified. This mechanism may influence the exposure of suckling infants to xenobiotics, as well as having potentially toxic effects on the lactating mammary epithelium and possibly altering the nutritional quality of the milk.

Is turtle longevity linked to enhanced mechanisms for surviving brain anoxia and reoxygenation?

We suggest that the processes that protect the turtle brain against anoxia and subsequent reoxygenation might also contribute to turtle longevity since many of them are linked to age related neurodegeneration. In the turtle the mechanisms for conserving ion channel function are particularly robust. The anoxic turtle brain avoids excitatory neurotransmitter toxicity by maintaining a balance between dopamine and glutamate-release and still active uptake mechanisms. In the anoxic turtle brain the inhibitory tone is strengthened through a sustained rise in extracellular GABA, and a corresponding increase in the density of GABA(A) receptors. The turtle has enhanced mechanisms that protect against the formation of ROS and mechanisms to protect from ROS damage. As many of these may be selectively activated during anoxia and recovery, the turtle could serve as a useful model to identify and investigate mechanisms for activating key protection and rescue mechanisms implicated in aging.

Uptake of 5-hydroxytryptamine by equine digital vein endothelial cells: inhibition by amines found in the equine caecum.

5-hydroxytryptamine (5-HT; serotonin) is a potent vasoconstrictor of equine digital blood vessels and has been implicated in the pathogenesis of acute laminitis. The aims of this study were firstly to examine whether cells of the digital blood vessel wall exhibited an active uptake mechanism for 5-HT and to characterise its efficiency; and secondly, to study the potential inhibitory effect on this process of other amines, produced in the equine caecum. Confluent monolayers of equine digital vein endothelial cells (EDVEC) and equine digital vein smooth muscle cells (EDVSMC) were incubated with [3H]5-HT (0.1-250 micromol/l) and the total and active uptake calculated. Equine pulmonary vein endothelial cells (EPVEC) were used as a positive control. Both EDVEC and EDVSMC showed uptake of [3H]5-HT by nonfaci litated diffusion; however, only EDVEC showed evidence of saturable facilitated uptake mechanism, with a Km of 41.6 +/- 9.3 micromol/l, which was significantly higher than that of EPVEC (9.9 +/- 2.1 micromol/l). All 6 caecally-derived amines examined (tyramine, spermine, isoamylamine, tryptamine, phenylethylamine and isobutylamine) inhibited the total uptake of [3H]5-HT in a concentration-dependent manner, tyramine having the lowest IC50 (3.7 x 10(-6) mol/l). These data suggest that facilitated uptake into the endothelium could play a role in moderating the vasoconstrictor response to 5-HT in the equine digital circulation. The vasoconstrictor action of 5-HT could be potentiated by gut-derived amines, providing a feasible link between GI disturbances and the pathophysiology of laminits.

Novel Mitochondrial Creatine Transport Activity: IMPLICATIONS FOR INTRACELLULAR CREATINE COMPARTMENTS AND BIOENERGETICS

Immunoblotting of isolated mitochondria from rat heart, liver, kidney, and brain with antibodies made against N- and C-terminal peptide sequences of the creatine transporter, together with in situ immunofluorescence staining and immunogold electron microscopy of adult rat myocardium, revealed two highly related polypeptides with molecular masses of approximately 70 and approximately 55 kDa in mitochondria. These polypeptides were localized by immunoblotting of inner and outer mitochondrial membrane fractions, as well as by immunogold labeling in the mitochondrial inner membrane. In addition, a novel creatine uptake via a mitochondrial creatine transport activity was demonstrated by [(14)C]creatine uptake studies with isolated mitochondria from rat liver, heart, and kidney showing a saturable low affinity creatine transporter, which was largely inhibited in a concentration-dependent manner by the sulfhydryl-modifying reagent NEM, as well as by the addition of the above anti-creatine transporter antibodies to partially permeabilized mitochondria. Mitochondrial creatine transport was to a significant part dependent on the energetic state of mitochondria and was inhibited by arginine, and to some extent also by lysine, but not by other creatine analogues and related compounds. The existence of an active creatine uptake mechanism in mitochondria indicates that not only creatine kinase isoenzymes, but also creatine transporters and thus a certain proportion of the creatine kinase substrates, might be subcellularly compartmentalized. Our data suggest that mitochondria, shown here to possess creatine transport activity, may harbor such a creatine/phosphocreatine pool.

Functional Characterization of Two Ripening-related Sucrose Transporters from Grape Berries

The ripening of grape (Vitis vinifera L.) berries is associated with a large accumulation of glucose and fructose in the vacuoles of the fruit cells. These hexoses are derived from sucrose, which is released from the phloem and may be taken up by parenchyma cells prior to hydrolysis. We have expressed two putative ripening-related sucrose transporters from grape berries, VvSUC11 (synonymous with VvSUT1) and VvSUC12, in an invertase deficient yeast strain to characterize their transport activities. Sucrose was taken up by yeast transformed with either transporter at an optimum pH of <4.5 and with a Michaelis constant (Km) of 0.9–1.4m M. The uptake of sucrose through VvSUC11 and VvSUC12 was inhibited by protonophores and by vanadate. This is consistent with an active uptake mechanism involving proton cotransport, typical of sucrose/H+symporters. The transporters from grape berries were functionally similar to Scr1, a sucrose transporter from Ricinus cotyledons. It is likely that in grape berries VvSUC11 and VvSUC12 facilitate the loading of sucrose from the apoplast into the parenchyma cells.

Evolutionary and ecological perspectives on carbon acquisition in phytoplankton

Statistically significant differences exist among the major algal and cyanobacterial taxa in the catalytic efficiency of Ribulose‐1,5 bisphosphate carboxylase/oxygenase (RubisCO)—the rate limiting carboxylase in C‐fixation. With the notable exception of the red algae, a comparison of the kinetic properties of RubisCO among phytoplankton of different geological ages suggests that the enzyme has evolved towards higher CO2 affinity in response to decreasing atmospheric CO2/O2 ratios over geological time. Taxonomic differences in the kinetic properties of RubisCO influence the mechanisms of C acquisition among phytoplankton groups. A significant negative correlation exists between the CO2 specificity factor of RubisCO and the capacity of cells to concentrate inorganic C through active uptake mechanisms. Differential resource requirements associated with C acquisition could affect the relative fitness of phytoplankton groups and thus their ecological interactions. I argue that inorganic C may play a more important role in phytoplankton productivity and community ecology than has been previously recognized and highlight some outstanding questions for future research.

Phosphorus and potassium uptake by woody roots of twelve-year-old slash pine trees

Under natural conditions, woody roots contribute over 80% of the fine-root system of slash pine ( Pinus elliottii Engelm. var. elliotii) and have the potential to play a major role in nutrient uptake of field grown trees. However, although nutrient uptake by `woody roots' of trees has been reported, these studies have used brown coloration to define woodiness of roots, despite color being a poor indicator of anatomical attributes that defines secondary growth. Nutrient uptake by brown roots has also generally been measured using excised roots or intact roots from tree seedlings grown under laboratory or greenhouse conditions and which may have little relevance to field conditions. This study documented P and K depletion of intact fine roots for 12-year old slash pines growing under field conditions. The objectives were: (i) to compare P and K depletion between whole root systems and woody root systems of slash pine ( Pinus elliotii Engelm. var. elliotii); and (ii) to compare P and K depletion between the whole-root system and woody-root systems when N 2 gas was used as a metabolic inhibitor of ion absorption. Woody-root components were isolated from whole-root systems (woody and non-woody) by waxing or cutting. N 2 gas was also used as a metabolic inhibitor of P and K depletion and compared to P and K depletion when air gas was used. We concluded that woody roots function in P and K uptake, and that an active uptake mechanism is implicated. Rates of P and K uptake for woody roots were similar per unit root length to rates of ion uptake of whole (woody and non-woody) roots.

Chapter 4 Interactions between the immune and neuroendocrine systems

The concept that the mind can affect the body, that emotions can affect disease, is not new. From before the time of Hippocrates until today, the popular culture has championed the belief that stress and the emotions can affect bodily disease. Until recently, however, this concept was dismissed by the scientific and medical community as unfounded. However, the availability of technological tools in neurobiology and immunology, and in the related clinical disciplines of clinical psychiatry, psychology, and rheumatology, has permitted definition of the molecular, biochemical and neuroanatomical pathways by which the immune system and the central nervous systems communicate. Through defining these communication networks, it is possible to better understand the diseases that result when these pathways are interrupted. Such understanding is leading not only to the scientific community's acceptance of the concept of wholeness of mind and body, but to the development of novel approaches to treat illnesses which result when such communication networks are disturbed. There are many levels and mechanisms by which the immune and central nervous system communicate, and interruptions or perturbations of each can alter the severity, course and susceptibility to, and resistance to inflammatory, autoimmune, and infectious diseases. The immune system signals the central nervous system at multiple levels and through many mechanisms, depending on the route of administration, dose and specific cytokine involved. Cytokines can signal the brain via systemic vascular routes, by crossing the blood brain barrier at leaky points (the circumventricular organs), or by specific active transport uptake mechanisms. One of the most important mechanisms by which cytokines signal the brain is through activation of second messenger synthesizing enzymes and resultant production of second messengers, such as the nitric oxide synthase (NOS)/nitric oxide and the cyclooxygenase/prostaglandin system.

Inhibition of cell growth by a fused protein of human ribonuclease 1 and human basic fibroblast growth factor.

Pancreatic-type RNases are considered to have cytotoxic potential due to their ability to degrade RNA molecules when they enter the cytosol. However, most of these RNases show little cytotoxicity because cells have no active uptake mechanism for these RNases and because the ubiquitous cytoplasmic RNase inhibitor is considered to play a protective role against the endocytotic leak of RNases from the outside of cells. To study the cytotoxic potential of RNase toward malignant cells targeting growth factor receptors, the C-terminus of human RNase 1 was fused to the N-terminus of human basic fibroblast growth factor (bFGF). This RNase-FGF fused protein effectively inhibited the growth of mouse melanoma cell line B16/BL6 with high levels of cell surface FGF receptor. This effect appeared to result from prolongation of the overall cell cycle rather than the killing of cells or specific arrest in a particular phase of the cell cycle. Thus, human RNase 1 fused to a ligand of cell surface molecules, such as the FGF receptor, is shown to be an effective candidate for a selective cell targeting agent with low toxic effects on normal cell types.

Amino acid sequence of proteins induced in Fe-deficient stressed alfalfa (Medicago sativa L.) roots

Alfalfa (Medicago sativa L.) grows well in soils with a moderately high pH and dissolves insoluble iron in the rhizosphere. We have investigated active uptake mechanisms under Fe-deficient nutrient conditions and the effects of Fe-deficiency on plants. Previously, we observed that Fe-deficient alfalfa roots exuded many compounds (Masaoka et al. 1993) such as fiavonoids. We also identified a new compound “alfafuran” which is a phenol compound and is different from organic acids or phytosiderophore-type amino acid derivatives exuded by Fe-deficient plant roots. This compound is also very effective in dissolving ferric phosphate (Noguchi et al. 1994), suggesting that alfalfa may have developed several strategies against Fe-deficient stress including the exudation of organic compounds like alfafuran which accelerate the Fe3+-reducing activity on the root cell membrane to dissolve insoluble iron compounds. Suzuki et al. (1995, 1997) observed that in barley several peptide spots obtained by electrophoresis were induced under Fe-deficient stress when mugineic acid-family phytosiderophores were secreted from the roots. They suggested that these peptides control the mugineic acid synthesis and secretion. We examined the peptides induced in Fe-deficient alfalfa roots.

Cationic polyhexylcyanoacrylate nanoparticles as carriers for antisense oligonucleotides.

After antisense oligodeoxynucleotides (ODNs) were suggested for therapeutic use in 1978, major advances were made in developing modified oligonucleotides with increased nuclease resistance and improved cellular uptake. In the present report, positively charged nanoparticles prepared from diethylaminoethyl (DEAE)-dextran and polyhexylcyanoacrylate (PHCA) were evaluated as carriers for ODNs. The oligonucleotides were analyzed by anion-exchange HPLC. The nanoparticles exhibited a high loading capacity, with approximately 35 mumol ODNs adsorbed per gram of polymeric material. The adsorption efficacy was found to be dependent on the pH, on the ionic strength of the medium, and on the amount of DEAE-dextran. Highest loading for ODNs was achieved at pH 5.5, using a 10 mM phosphate buffer. Oligonucleotides adsorbed to the surface of the nanoparticles were nearly completely protected against degradation by the endonuclease DNase I and under in vitro cell culture conditions, whereas unprotected ODNs were totally digested under these conditions. Nanoparticles led to a 20-fold increase in cellular uptake of FITC-oligonucleotides. The internalized oligonucleotides were frequently localized as vesicular structures in the cytoplasmatic compartment. Because of their temperature-dependent uptake, we propose an active uptake mechanism, such as endocytosis, for the internalization of the ODN-nanoparticle formulations.