Evaluation of molecule-microbe interactions with capillary electrophoresis: procedures, utility and restrictions

Understanding the interactions between molecules and living organisms is of paramount importance for the evaluation of pharmaceutical activity, chemical toxicity and all manner of microbiological studies. The capability of capillary electrophoresis (CE) in the evaluation of molecule-microbe interactions is examined in the present paper. The fundamental chemical concept of the binding or association constant for molecular systems measured in free solution is discussed for biological systems where microorganisms uptake or associate with molecules from their environment. The heterogeneity of the living organisms must be understood and accounted for including differences related to semantics such as concentration units and the nature of the associations between two entities and large differences in the size and number of microorganisms as compared to molecules. Finally, the added complexity and even inhomogeneity of a cell compared to most molecular systems must be considered and possibly controlled. The binding of specific molecules to viruses is discussed. CE can be utilized to quickly determine if a molecule binds very strongly or not at all to a cell (i.e., a binary yes/no answer). This could be useful for initial high-throughput screening purposes when using capillary arrays, for example. CE can be useful for determining unusual (large) molecule/microbe stoichiometries. Finally, CE can sometimes be used to determine the size of binding constants (K(RL)) within certain limits provided experimental conditions can be formulated that minimize problems of biological heterogeneity.     

A novel general route for the preparation of enantiopure imidazolines

A novel procedure for the preparation of enantiopure 1,4-disubstituted 2-imidazolines is reported. Enantiopure beta-amino alcohols are converted into N-hydroxyethylamides, which are reacted with excess thionyl chloride, or with thionyl chloride followed by phosphorus pentachloride to yield N-chloroethylimidoyl chlorides. These intermediates are treated with amines and anilines to produce N-chloroethylamidines, which are converted into imidazolines upon workup with aqueous hydroxide. The method is simple and efficient and has been used to prepare a wide variety of enantiopure imidazolines, in a modular fashion, from readily available amino alcohols.     

The In Vitro Anti-pathogenic Activity of Immunoglobulin Concentrates Extracted from Ovine Blood

An immunoglobulin-rich fraction has been prepared from ovine blood in our laboratory. We have investigated its antibacterial activity and binding activity to pathogenic whole cell antigens, lipopolysaccharide (LPS) and staphylococcal enterotoxin B. Ovine immunoglobulin concentrate (OIC) comprised about 73 ± 2% of IgG and 11 ± 1% of IgM on a protein basis. It inhibited the growth of all 13 strains of pathogens tested, but the inhibitory activity varied according to bacterial strain. The inhibitory activity of OIC was attributed to the high contents of undenatured immunoglobulin present because its inhibitory activity was destroyed by pepsin digestion and heat treatment (65°C for 30 min). OIC bound to all the Gram-positive and Gram-negative pathogens, regardless of cell wall structure. The highest magnitude of crossreactivity to whole cell antigens was against Staphylococcus epidermidis and Shigella soneii strains (p < 0.001). The binding activity of OIC to LPS obtained from Escherichia coli O111:B4 and Salmonella enterica serotype typhimurium was assessed by enzyme-linked immunosorbent assay and lymphoblast K-562 proliferation assay. OIC bound to LPS with a binding activity that was dependent on OIC concentration and saturable, showing typical hyperbolic curves. For toxin-binding activity, an OIC concentration-dependent trend like that for LPS-binding activity was also observed. This preliminary evidence suggests that the OIC used in this study could be a promising supplement for protecting against pathogenic bacteria.     

Cellulose Iβ investigated by IR-spectroscopy at low temperatures

Highly crystalline oriented Halocynthia roretzi cellulose Iβ films were investigated by IR-spectroscopy between ?180 and +10 °C. Changes in the IR-spectra induced by temperature were compared to published changes induced by mechanical stretching. This made it possible to conclude that frequency shifts in the O–H stretching region of the IR-spectra due to temperature were not predominantly an indirect effect of thermal expansion leading to greater O–O distances, but were due directly to the effect of temperature on the O–H···O hydrogen bonds. Temperature induced frequency shifts of C–H stretching bands were consistent with the presence of weak inter-sheet C–H···O bonds. Furthermore, no phase transition in cellulose Iβ was found between ?180 and +10 °C.     

Communication: An Unusual Side Reaction in the Perallylation of a Glucoside Brought About by Sodium Hydride

We are presently working on the combination of carbohydrate and dendrimer chemistry, both to develop the synthesis of multivalent glycomimetics and to prepare novel dendrimers with advantageous properties. In the course of this work we have used saccharides as oligofunctional core molecules for the synthesis of carbohydrate-centered dendrimers¹ and carbohydrate-centered glycoclusters.²     

Evaluation of sphingomyelin,cholester, and phosphatidylcholine-based immobilized artificial membrane liquid chromatography to predict drug penetration across the blood-brain barrier

Over the past decades, several in vitro methods have been tested for their ability to predict drug penetration across the blood-brain barrier. So far, in high-performance liquid chromatography, most attention has been paid to micellar liquid chromatography and immobilized artificial membrane (IAM) LC. IAMLC has been described as a viable approach, since the stationary phase emulates the lipid environment of a cell membrane. However, research in IAMLC has almost exclusively been limited to phosphatidylcholine (PC)-based stationary phases, even though PC is only one of the lipids present in cell membranes. In this article, sphingomyelin and cholester stationary phases have been tested for the first time towards their ability to predict drug penetration across the blood-brain barrier. Upon comparison with the PC stationary phase, the sphingomyelin- and cholester-based columns depict similar predictive performance. Combining data from the different stationary phases did not lead to improvements of the models. Figure

Schematic representation of how IAM-LC is used to predict drug penetration across the blood-brain barrier.     

A framework for the automated real-time detection of short tonal sounds from ocean observatories

Acquisition of acoustic data from ocean observatories is expected to play a key role for the long-term monitoring of marine mammals and anthropogenic noise. It typically requires processing of a large volume of acoustic data and it must rely on automated identification of signals. We present an algorithmic framework for the detection of short tonal sounds (e.g. cetacean calls, anthropogenic pings) intended to act as a first stage in a system for the automated real-time detection, classification, and localisation of acoustic sources. The algorithm was validated under a diversity of scenarios expected at ocean observatories. Using simulated signals that emulate a variety of cetacean call-types, perfect identification of signal position was obtained for signal to noise ratios of ?15 to ?5 dB, depending on the signal-type. Separation of real-world data segments with short tonal sounds (mainly cetacean calls) from segments with other sounds or noise resulted in Area Under the ROC Curve values between 0.96 and 0.98. The algorithm can be used to automatically identify cetacean calls and anthropogenic short tonal sounds much faster than in real-time, thereby reducing the burden put on data transmission, storage, or processing by classification and localisation algorithms.     

Ultrafast excited state dynamics controlling photochemical isomerization of N-methyl-4-[trans-2-(4-pyridyl)ethenyl]pyridinium coordinated to a {Re I(CO)3(2,2'-bipyridine)} chromophore

Two multifunctional photoactive complexes [Re(Cl)(CO)(3)(MeDpe(+))(2)](2+) and [Re(MeDpe(+))(CO)(3)(bpy)](2+) (MeDpe(+)=N-methyl-4-[trans-2-(4-pyridyl)ethenyl]pyridinium, bpy=2,2'-bipyridine) were synthesized, characterized, and their redox and photonic properties were investigated by cyclic voltammetry; ultraviolet-visible-infrared (UV/Vis/IR) spectroelectrochemistry, stationary UV/Vis and resonance Raman spectroscopy; photolysis; picosecond time-resolved absorption spectroscopy in the visible and infrared regions; and time-resolved resonance Raman spectroscopy. The first reduction step of either complex occurs at about -1.1 V versus Fc/Fc(+) and is localized at MeDpe(+). Reduction alone does not induce a trans-->cis isomerization of MeDpe(+). [Re(Cl)(CO)(3)(MeDpe(+))(2)](2+) is photostable, while [Re(MeDpe(+))(CO)(3)(bpy)](2+) and free MeDpe(+) isomerize under near-UV irradiation. The lowest excited state of [Re(Cl)(CO)(3)(MeDpe(+))(2)](2+) has been identified as the Re(Cl)(CO)(3)-->MeDpe(+ 3)MLCT (MLCT=metal-to-ligand charge transfer), decaying directly to the ground state with lifetimes of approximately 42 (73 %) and approximately 430 ps (27 %). Optical excitation of [Re(MeDpe(+))(CO)(3)(bpy)](2+) leads to population of Re(CO)(3)-->MeDpe(+) and Re(CO)(3)-->bpy (3)MLCT states, from which a MeDpe(+) localized intraligand (3)pipi* excited state ((3)IL) is populated with lifetimes of approximately 0.6 and approximately 10 ps, respectively. The (3)IL state undergoes a approximately 21 ps internal rotation, which eventually produces the cis isomer on a much longer timescale. The different excited-state behavior of the two complexes and the absence of thermodynamically favorable interligand electron transfer in excited [Re(MeDpe(+))(CO)(3)(bpy)](2+) reflect the fine energetic balance between excited states of different orbital origin, which can be tuned by subtle structural variations. The complex [Re(MeDpe(+))(CO)(3)(bpy)](2+) emerges as a prototypical, multifunctional species with complementary redox and photonic behavior.