Peptide-based strategies for enhanced cell uptake, transcellular transport, and circulation: Mechanisms and challenges.

Peptides are emerging as a new tool in drug and gene delivery. Peptide-drug conjugates and peptide-modified drug delivery systems provide new opportunities to avoid macrophage recognition and subsequent phagocytosis, cross endothelial and epithelial barriers, and enter the cytoplasm of target cells. Peptides are relatively small, low-cost, and are stable in a wide range of biological conditions. In this review, we summarize recent work in designing peptides to enhance penetration of biological barriers, increase cell uptake, and avoid the immune system. We highlight recent successes and contradictory results, and outline common emerging concepts and design rules. The development of sequence-structure-function relationships and standard protocols for benchmarking will be a key to progress in the field.

Evidence for NO(x) control over nighttime SOA formation.

Laboratory studies have established a number of chemical pathways by which nitrogen oxides (NO(x)) affect atmospheric organic aerosol (OA) production. However, these effects have not been directly observed in ambient OA. We report measurements of particulate organic nitrates in Bakersfield, California, the nighttime formation of which increases with NO(x) and is suppressed by high concentrations of organic molecules that rapidly react with nitrate radical (NO(3))--evidence that multigenerational chemistry is responsible for organic nitrate aerosol production. This class of molecules represents about a third of the nighttime increase in OA, suggesting that most nighttime secondary OA is due to the NO(3) product of anthropogenic NO(x) emissions. Consequently, reductions in NO(x) emissions should reduce the concentration of organic aerosol in Bakersfield and the surrounding region.

Measurement and prediction of the rate and extent of drug delivery into and through the skin.

Skin diseases are prevalent and can significantly affect quality of life. Empirical mathematical models retrospectively analyse data to predict skin permeation from the physico-chemical properties of drugs. Quantitative structure permeability relationships are discussed, along with alternatives to linear modelling. Mechanistic mathematical models derived from first principles are also considered. Further, in vitro experiments allow predictions to be made using suitable membranes (cultured cell lines or excised skins). In vivo methods to assess (trans)dermal drug delivery aim to minimise clinical studies, especially to determine whether formulations are bioequivalent. Microdialysis is discussed, together with the FDA-approved skin blanching (pharmacodynamic) assay for corticosteroids. The progress made with the tape stripping methodology is reviewed. Two distinct strategies have emerged, the first where the total amount of drug in the stratum corneum (SC) at one uptake and one clearance time are compared; and the second which generates drug permeation profiles across the SC, and allows dermatopharmacokinetic parameters to be derived.

X chromosome loss and ageing.

The detection of a low level 45,X cell line during routine cytogenetic analysis in an adult female can be difficult to interpret. In the absence of recent information regarding loss of the X chromosome and ageing, we undertook a prospective study. A total of 19,650 cells from 655 females aged from birth to 80 years were screened cytogenetically. The frequency of X chromosome loss ranged from 0.07% at age <16 years to 7.3% at >65 years of age and showed a highly significant quadratic relationship between X chromosome loss and ageing (P < or = 0.00001). We have produced a graphic representation that provides a minimum baseline age-related rate of X chromosome loss. This should assist diagnostic cytogenetics laboratories to determine the significance of 45,X cell lines detected in women of all ages. We also compared the frequency of 45,X cells in women referred with at least one spontaneous abortion with those referred for other reasons and found no significant difference. Thus, in our population, an excess of 45,X cells is not associated with pregnancy loss.

Organic and elemental carbon measurements during ACE-Asia suggest a longer atmospheric lifetime for elemental carbon.

During the ACE-Asia intensive field campaign (March 14-April 20, 2001), PM1.0 organic (OC) and elemental carbon (EC) concentrations were measured onboard the NOAA R/V Ronald H. Brown over the Northwest Pacific Ocean using a semi-continuous automated carbon analyzer downstream of a carbon-impregnated filter denuder. This OC and EC measurement achieved a mean time resolution of about 200 min over the Pacific Ocean, substantially lower than that achieved previously (24 h). The semi-continuous measurements, in which the adsorption artifact was substantially reduced using the denuder, showed good agreement with integrated artifact-corrected measurements made without a denuder. Mean particulate OC and EC concentrations were 0.21 and 0.09, 0.70 and 0.29, 1.00 and 0.27, and 2.43 and 0.66 microg of C m(-3) over the background Pacific Ocean, Asian-influenced Pacific Ocean, offshore of Japan, and Sea of Japan, respectively. On April 11, 90-min average OC and EC concentrations peaked at 4.0 and 1.3 microg of C m(-3), respectively, offshore of Korea over the Sea of Japan. The OC/EC ratio of 3.7 over the Sea of Japan and offshore of Japan was substantially higher than that of 2.5 over the Asian-influenced Pacific Ocean, even though backward air mass trajectories put the "Asian-influenced Pacific Ocean" sample downwind. The OC/EC ratio decreased with increasing time since the air mass encountered the source regions of China, Japan, and Korea. This suggests a longer atmospheric residence time for EC than for OC.

Highly toxinogenic but avirulent Park-Williams 8 strain of Corynebacterium diphtheriae does not produce siderophore.

The highly toxinogenic Park-Williams 8 strain of Corynebacterium diphtheriae grows slowly in vitro and is avirulent. C. diphtheriae Park-Williams 8 is defective in iron uptake and does not produce the corynebacterial siderophore corynebactin. Addition of partially purified corynebactin stimulated iron uptake and growth of iron-deprived C. diphtheriae Park-Williams 8 cells.

Genetic and biochemical evidence for a siderophore-dependent iron transport system in Corynebacterium diphtheriae.

During growth under conditions of iron deprivation, Corynebacterium diphtheriae secreted a siderophore into the culture medium. This extracellular siderophore was necessary for rates of iron uptake at pH 8.0 by C. diphtheriae C7 and related strains. We isolated a mutant of C. diphtheriae C7(beta), strain HC6, which did not make the corynebacterial siderophore. Strain HC6 grew very poorly, even under high-iron conditions, and had a severe defect in iron transport. Both growth and iron uptake by strain HC6 were greatly stimulated by the corynebacterial siderophore. We used strain HC6 to develop a bioassay for the corynebacterial siderophore and to look for other potential siderophores for C. diphtheriae. Among the purified phenolate and hydroxamate siderophores tested, only aerobactin was able to stimulate the growth of strain HC6. Partial purification of the corynebacterial siderophore was achieved. The siderophore did not give positive reactions in the Arnow test for phenolates or the Csaky test for hydroxamates and may have a novel chemical structure.

Initial characterization of the ferric iron transport system of Corynebacterium diphtheriae.

Transport of ferric iron into Corynebacterium diphtheriae C7(beta) was shown to occur by a high-affinity, active transport system. Optimal rates were at pH 6.8 and 40 degrees C. Strong inhibition of uptake by carbonyl cyanide m-chlorophenylhydrazone was consistent with the electrochemical proton gradient as the major energy source for iron transport, and inhibition by Hg2+ indicated that sulfhydryl groups were also important. Evidence was obtained for stimulation of iron uptake at pH 8.0 by a dialyzable, extracellular factor present in conditioned medium from low-iron cultures of C. diphtheriae.

Regulation of toxinogenesis in Corynebacterium diphtheriae: mutations in the bacterial genome that alter the effects of iron on toxin production.

Mutants of Corynebacterium diphtheriae C7(beta) that are resistant to the inhibitory effects of iron on toxinogenesis were identified by their ability to form colonies surrounded by toxin-antitoxin halos on agar medium containing both antitoxin and a high concentration of iron. Chromosomal mutations were essential for the altered phenotypes of four independently isolated mutant strains. During growth in deferrated liquid medium containing various amounts of added iron, these mutants differed from wild-type C. diphtheriae C7(beta) in several ways. Their growth rates were slower under low-iron conditions and were stimulated to various degrees under high-iron conditions. The concentrations of iron at which optimal toxin production occurred were higher for the mutants than for wild-type C. diphtheriae C7(beta). Toxin production by the mutants during growth in low-iron medium occurred throughout the period of exponential growth at nearly constant rates that were proportional to the bacterial growth rates. In contrast, toxin production by wild-type C. diphtheriae C7(beta) in similar low-iron cultures occurred predominantly during the late exponential phase, when iron was a growth-limiting nutrient. Additional studies demonstrated that these mutants had severe defects in their transport systems for ferric iron. We propose that the altered regulation of toxinogenesis by iron in our mutants was caused by the severe defects in their iron transport systems. As a consequence, the mutants exhibited a low-iron phenotype during growth under conditions that permitted wild-type C. diphtheriae C7(beta) to exhibit a high-iron phenotype.

Phosphate exchange in the pit transport system in Escherichia coli.

The Pit system of phosphate transport in Escherichia coli catalyzes a rapid exchange between the external inorganic phosphate and internal phosphate pools, including some ester phosphates which are in rapid equilibrium with the internal Pi pool. Unlike net energized uptake, the Pi exchange proceeds in energy-depleted cells in the presence of uncouplers and is not accompanied by the movement of potassium ions. In the absence of externally added phosphate, the exit of Pi from the cells is insignificant. The apparent Km for external Pi in the exchange reaction is about 7 mM (2 orders of magnitude higher than that of energized uptake), but the maximal velocity is about the same. The exchange is temperature sensitive and is affected by thiol reagents. The combined observations suggest the operation of a facilitator which is part of the Pit system. The exchange is repressed in cells grown on glucose and other phosphotransferase system substrates, but not in cells grown on other carbohydrate sources. The repression can be reversed by the addition of cyclic AMP to the medium.

The nature of the link between potassium transport and phosphate transport in Escherichia coli.

A series of mutants of Escherichia coli, combining defects in either of the two phosphate transport systems with defects in one or more of the potassium transport systems, was used to study the nature of the previously observed obligatory requirement for each one of these ions in the transport of the other. The results show that no pair of systems is obligatorily linked, and that either ion can be transported by any one of its systems, provided that a means of entry for the other ion is available. Furthermore, in the total absence of Pi, K+ entry accompanies the transport of other anions, such as aspartate, glutamate, sn-glycero-3-phosphate and glucose 6-phosphate. The results indicate that Pi and the other anions enter by symport with protons, and that a simultaneous K+/H+ exchange, which would serve to maintain the intracellular pH, is responsible for the observed K+ 'symport' with these anions.

Linked transport of phosphate, potassium ions and protons in Escherichia coli.

Pi entry into Escherichia coli cells through either of the two Pi-transport systems (Pit or Pst) prompts the influx of K+ and H+ in a ratio that depends on the external pH. The entry of Pi is absolutely dependent on the presence of K+, and the entry of K+ is equally dependent on the presence of Pi. Experiments with a number of mutants carrying any one functional Pi-transport system and one or more of the individual K+-transport systems indicate a permissive type of linkage of the two transports, in that there is no obvious preference by any of the Pi-transport systems for a particular K+-transport system for the concomitant entry of the two ions.

The role of calcium ions in the regulation of rat thymocyte pyruvate oxidation by mitogens.

1. Calcium concentrations in the nanomolar range cause a specific stimulation of the oxidation of pyruvate by isolated mitochondria from rat thymus that is sufficient to account precisely for the stimulation of pyruvate oxidation observed when rat thymocytes are incubated with the mitogens concanavalin A or ionophore A23187. 2. Higher concentrations of Ca2+ (more than 50 nM) inhibit the oxidation of NAD+-linked substrates by rat thymus mitochondria without affecting the oxidation of succinate or ascorbate+ NNN'N'-tetramethyl-p-phenylendiamine. 3. The addition of Ni2+ or Co2+ (2mM) to rat thymocytes prevents the response to concanavalin A at the level of pyruvate oxidation without affecting the stimulation of glycolysis induced by this mitogen. In contrast, the complete metabolic response to the ionophore A23187 is abolished by these ions. Ni2+ and Co2+ interfere with the ability of the ionophore to transport Ca2+ across the plasma membrane. 4. Concanavalin A, but not ionophore A23187, increases the respiratory inhibition induced by Ni2+ and Co2+. 5. These results support the view that mitogens stimulate lymphocyte pyruvate oxidation through an increase in cellular Ca2+ uptake.