Capillary electrochromatography and quartz crystal microbalance, valuable techniques in the study of heparin-lipoprotein interactions

Atherosclerosis is initiated when lipoproteins bind to proteoglycans (PGs) in arterial walls. The binding is mediated by apolipoprotein apoB-100 and/or apoE, both of which have binding affinity toward heparin. We developed covalently bound heparin coatings for APTES-modified silica capillaries and SiO(2) chips and carried out capillary electrochromatography (CEC) and quartz crystal microbalance (QCM) studies on the interactions of heparin with selected peptide fragments of apoB-100 and apoE and, for CEC, also with low- and high-density lipoproteins (LDL and HDL), the latter with and without apoE. The peptides are known to mediate interactions of HDL and LDL with arterial PGs. Interactions and affinities were expressed in CEC as retention factors and reduced mobilities and in continuous flow QCM techniques as affinity constants. Both techniques showed heparin interactions to be stronger with apoB-100 peptide than with apoE peptide fragment, and they confirmed that the sulfate groups in heparin play an especially important role in interactions with apoB-100 peptide fragments. In addition, CEC confirmed the importance of sulfate groups of heparin in interactions between heparin and LDL and between heparin and apoE-containing HDL. CEC and QCM acted as excellent platforms to mimic these biologically important interactions, with small sample and reagent consumption.     

Comparison of rat epidermal keratinocyte organotypic culture (ROC) with intact human skin: Lipid composition and thermal phase behavior of the stratum corneum

The present report is a part of our continuing efforts to explore the utility of the rat epidermal keratinocyte organotypic culture (ROC) as an alternative model to human skin in transdermal drug delivery and skin irritation studies of new chemical entities and formulations. The aim of the present study was to compare the stratum corneum lipid content of ROC with the corresponding material from human skin. The lipid composition was determined by thin-layer chromatography (TLC) and mass-spectrometry, and the thermal phase transitions of stratum corneum were studied by differential scanning calorimetry (DSC). All major lipid classes of the stratum corneum were present in ROC in a similar ratio as found in human stratum corneum. Compared to human skin, the level of non-hydroxyacid-sphingosine ceramide (NS) was increased in ROC, while α-hydroxyacid-phytosphingosine ceramide (AP) and non-hydroxyacid-phytosphingosine ceramides (NP) were absent. Also some alterations in fatty acid profiles of ROC ceramides were noted, e.g., esterified ω-hydroxyacid-sphingosine contained increased levels of oleic acid instead of linoleic acid. The fraction of lipids covalently bound to corneocyte proteins was distinctly lower in ROC compared to human skin, in agreement with the results from DSC. ROC underwent a lipid lamellar order to disorder transition (T₂) at a slightly lower temperature (68 °C) than human skin (74 °C). These differences in stratum corneum lipid composition and the thermal phase transitions may explain the minor differences previously observed in drug permeation between ROC and human skin.     

Vitamin C enhances differentiation of a continuous keratinocyte cell line (REK) into epidermis with normal stratum corneum ultrastructure and functional permeability barrier

A continuous rat epidermal cell line (rat epidermal keratinocyte; REK) formed a morphologically well-organized epidermis in the absence of feeder cells when grown for 3 weeks on a collagen gel in culture inserts at an air-liquid interface, and developed a permeability barrier resembling that of human skin. By 2 weeks, an orthokeratinized epidermis evolved with the suprabasal layers exhibiting the differentiation markers keratin 10, involucrin, and filaggrin. Granular cells with keratohyalin granules and lamellar bodies, and corneocytes with cornified envelopes and tightly packed keratin filaments were present. Morphologically, vitamin C supplementation of the culture further enhanced the normal wavy pattern of the stratum corneum, the number of keratohyalin granules present, and the quantity and organization of intercellular lipid lamellae in the interstices of the stratum corneum. The morphological enhancements observed with vitamin C correlated with improved epidermal barrier function, as indicated by reduction of the permeation rates of tritiated corticosterone and mannitol, and transepidermal water loss, with values close to those of human skin. Moreover, filaggrin mRNA was increased by vitamin C, and western blots confirmed higher levels of profilaggrin and filaggrin, suggesting that vitamin C also influences keratinocyte differentiation in aspects other than the synthesis and organization of barrier lipids. The unique REK cell line in organotypic culture thus provides an easily maintained and reproducible model for studies on epidermal differentiation and transepidermal permeation.     

Natural hierarchy emerges from energy dispersal

Hierarchical organization of ‘systems within systems’ is an apparent characteristic of nature. For many biotic and abiotic systems it is known how the nested structural and functional order builds up, yet the general principle why matter evolves to hierarchies has remained unfamiliar to many. We clarify that increasingly larger integrated systems result from the quest to decrease free energy according to the 2nd law of thermodynamics. The argumentation is based on the recently derived equation of motion for natural processes. Hierarchically organized energy transduction machinery emerges naturally when it provides increased rates of energy dispersal. Likewise, a hierarchical system will dismantle into its constituents when they as independent systems will provide higher rates of entropy increase. Since energy flows via interactions, decreasing strengths of interactions over increasingly larges lengths scales mark natural boundaries for nested integrated systems.     

Identification of a novel streptococcal adhesin P (SadP) protein recognizing galactosyl-α1-4-galactose-containing glycoconjugates: convergent evolution of bacterial pathogens to binding of the same host receptor

Bacterial adhesion is often a prerequisite for infection, and host cell surface carbohydrates play a major role as adhesion receptors. Streptococci are a leading cause of infectious diseases. However, only few carbohydrate-specific streptococcal adhesins are known. Streptococcus suis is an important pig pathogen and a zoonotic agent causing meningitis in pigs and humans. In this study, we have identified an adhesin that mediates the binding of S. suis to galactosyl-α1-4-galactose (Galα1-4Gal)-containing host receptors. A functionally unknown S. suis cell wall protein (SSU0253), designated here as SadP (streptococcal adhesin P), was identified using a Galα1-4Gal-containing affinity matrix and LC-ESI mass spectrometry. Although the function of the protein was not previously known, it was recently identified as an immunogenic cell wall protein in a proteomic study. Insertional inactivation of the sadP gene abolished S. suis Galα1-4Gal-dependent binding. The adhesin gene sadP was cloned and expressed in Escherichia coli. Characterization of its binding specificity showed that SadP recognizes Galα1-4Gal-oligosaccharides and binds its natural glycolipid receptor, GbO(3) (CD77). The N terminus of SadP was shown to contain a Galα1-Gal-binding site and not to have apparent sequence similarity to other bacterial adhesins, including the E. coli P fimbrial adhesins, or to E. coli verotoxin or Pseudomonas aeruginosa lectin I also recognizing the same Galα1-4Gal disaccharide. The SadP and E. coli P adhesins represent a unique example of convergent evolution toward binding to the same host receptor structure.     

Phenolic Acid Composition,Antiatherogenic and Anticancer Potential of Honeys Derived from Various Regions in Greece

The phenolic acid profile of honey depends greatly on its botanical and geographical origin. In this study, we carried out a quantitative analysis of phenolic acids in the ethyl acetate extract of 12 honeys collected from various regions in Greece. Our findings indicate that protocatechuic acid, p-hydroxybenzoic acid, vanillic acid, caffeic acid and p-coumaric acid are the major phenolic acids of the honeys examined. Conifer tree honey (from pine and fir) contained significantly higher concentrations of protocatechuic and caffeic acid (mean: 6640 and 397 µg/kg honey respectively) than thyme and citrus honey (mean of protocatechuic and caffeic acid: 437.6 and 116 µg/kg honey respectively). p-Hydroxybenzoic acid was the dominant compound in thyme honeys (mean: 1252.5 µg/kg honey). We further examined the antioxidant potential (ORAC assay) of the extracts, their ability to influence viability of prostate cancer (PC-3) and breast cancer (MCF-7) cells as well as their lowering effect on TNF- α-induced adhesion molecule expression in endothelial cells (HAEC). ORAC values of Greek honeys ranged from 415 to 2129 µmol Trolox equivalent/kg honey and correlated significantly with their content in protocatechuic acid (p<0.001), p-hydroxybenzoic acid (p<0.01), vanillic acid (p<0.05), caffeic acid (p<0.01), p-coumaric acid (p<0.001) and their total phenolic content (p<0.001). Honey extracts reduced significantly the viability of PC-3 and MCF-7 cells as well as the expression of adhesion molecules in HAEC. Importantly, vanillic acid content correlated significantly with anticancer activity in PC-3 and MCF-7 cells (p<0.01, p<0.05 respectively). Protocatechuic acid, vanillic acid and total phenolic content correlated significantly with the inhibition of VCAM-1 expression (p<0.05, p<0.05 and p<0.01 respectively). In conclusion, Greek honeys are rich in phenolic acids, in particular protocatechuic and p-hydroxybenzoic acid and exhibit significant antioxidant, anticancer and antiatherogenic activities which may be attributed, at least in part, to their phenolic acid content.     

Nocturnal drift of mayfly nymphs as a post-contact antipredator mechanism

1. The predominantly nocturnal constrained drift of stream invertebrates is commonly regarded as a behaviour that avoids encounters with visually foraging fish in the water column. The alternative explanation, that drift peaks are caused by bottom-feeding, nocturnal predators, has rarely been tested.
2. We examined these hypotheses by collecting invertebrate drift in five streams in northern Finland: one with brown trout ( Salmo trutta , a drift-feeding fish), one with alpine bullhead ( Cottus poecilopus , a benthic fish), one with both species, and two fishless streams.
3. Drift by Baetis mayflies was aperiodic or slightly diurnal in both fishless streams on all sampling occasions. In contrast, drift was nocturnal in streams with trout and, to a lesser extent, in the stream with bullhead. Non-dipteran prey drifted mainly nocturnally in all streams with fish, whereas Diptera larvae were less responsive to the presence of fish.
4. In laboratory experiments, bullheads were night-active, causing a much higher frequency of drift by touching Baetis at night than during the day. Thus, increased nocturnal drift may serve to avoid both visual predators (a pre-contact response) and benthic fish (a post-contact response). In streams with bottom-feeding fish, nocturnal drift should be caused by increased drift by night rather than by reduced drift by day.