Brain and Spinal Cord Distribution of Biphalin: Correlation with Opioid Receptor Density and Mechanism of CNS Entry

Abstract: Biphalin [(Tyr-d -Ala-Gly-Phe-NH)₂] is a bivalent, opioid peptide containing two pharmacophores linked by a hydrazine bridge. When administered intracerebroventricularly, it has been shown to be more potent than morphine and etorphine at eliciting antinociception. Biphalin has also been shown to cross both the blood-brain and blood-cerebrospinal fluid barriers. To understand the basis of biphalin's potency, regional brain and spinal cord distribution studies with [¹²⁵I-Tyr¹]biphalin were performed 5, 20, and 40 min after intravenous bolus injections. A statistically greater amount of [¹²⁵I-Tyr¹]-biphalin was detected in the nucleus accumbens compared with other brain regions (p < 0.05). This correlates with the high density of δ- and μ-opioid receptor mRNA and binding sites shown to be expressed in the nucleus accumbens. Also, a statistically greater amount of [¹²⁵I-Tyr¹]biphalin was detected in two other circumventricular organs, the choroid plexus and pituitary, when compared with other brain regions. These studies provide evidence that biphalin can reach not only brain sites, but also spinal sites to elicit antinociception. The overall CNS distribution of [¹²⁵I-Tyr¹]biphalin was decreased with naloxone, d -Phe-Cys-Tyr-d -Trp-Arg-Thr-Pen-Thr-NH₂, or naltrindole pretreatment, showing that biphalin detected in the brain and spinal cord is binding to δ- and μ-opioid receptors. Additional in situ brain perfusion experiments identified a saturable component contributing to CNS entry of [¹²⁵I-Tyr¹]biphalin, which could be described by Michaelis-Menten kinetics with a K_m of 2.6 ± 4.8 µM, V_max of 14.6 ± 2.89 pmol^?1·min^?1·g^?1, and K_d of 0.568 ± 0.157 µl·min^?1·g^?1. Brain entry of [¹²⁵I-Tyr¹]biphalin was sensitive to 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid and l -phenylalanine, suggesting use of the large neutral amino acid carrier. This work provides evidence that biphalin is a promising, potent analgesic that has a unique mechanism for reaching both spinal and supraspinal opioid receptor sites.     

Electron paramagnetic resonance saturation studies of P-700 reaction center chlorophyll in plant photosynthesis

Electron paramagnetic resonance (EPR) power saturation and saturation recovery methods have been used to determine the spin lattice, T₁, and spin-spin, T₂, relaxation times of P-700⁺ reaction-center chlorophyll in Photosystem I of plant chloroplasts for 10 K T 100 K. T₁ was 200 μs at 100 K and increased to 900 μs at 10 K. T₂ was 40 ns at 40 K and increased to 100 ns at 10 K. T₁ for 40 K T 100 K is inversely proportional to temperature, which is evidence of a direct-lattice relaxation process. At T = 20 K, T₁ deviates from the 1/T dependence, indicating a cross relaxation process with an unidentified paramagnetic species. The individual effects of ascorbate and ferricyanide on T₁ of P-700⁺ were examined: T₁ of P-700⁺ was not affected by adding 10 mM ascorbate to digitonin-treated chloroplast fragments (D144 fragments). The P-700⁺ relaxation time in broken chloroplasts treated with 10 mM ferricyanide was 4-times shorter than in the untreated control at 40 K. Ferricyanide appears to be relaxing the P-700⁺ indirectly to the lattice by a cross-relaxation process. The possibility of dipolar-spin broadening of P-700⁺ due to either the iron-sulfur center A or plastocyanin was examined by determining the spin-packet linewidth for P-700⁺ when center A and plastocyanin were in either the reduced or oxidized states. Neither reduced center A nor oxidized plastocyanin was capable of broadening the spin-packet linewidth of the P-700⁺ signal. The absence of diplolar broadening indicates that both center A and plastocyanin are located at a distance at least 3.0 nm from the P-700⁺ reaction center chlorophyll. This evidence supports previous hypotheses that the electron donor and acceptor to P-700 are situated on opposite sides of the chloroplast membrane. It is also shown that the ratio of photo-oxidized P-700 to photoreduced centers A and B at low temperature is 2 : 1 if P-700 is monitored at a nonsaturating microwave power.     

Effects of temperature,lipid modification and pH on the mobility of the major proteins of the receptor-rich membranes from Torpedo marmorata

The factors influencing the overall mobility of the major proteins of the acetylcholine receptor-rich membranes from Torpedo marmorata have been investigated by saturation transfer ESR spectroscopy and the lateral distribution of these proteins has been studied by electron microscopy. A spin-labelled derivative of maleimide, 3-maleimido-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (MSL), was used under various conditions of incubation, enabling us to attach it mainly to either an extrinsic protein of 43 kdaltons, or an intrinsic protein (40 kdaltons) bearing the α-toxin-binding site. (1) The direct reaction of MSL with the membrane fragments resulted in almost exclusive labelling of the 43 kdalton protein, an extrinsic protein located on the inner face of the receptor-rich membranes. (2) After the free SH groups were blocked with $\text{N-}\text{ethylmaleimide}$ and the disulfide bridges opened with the reducing agent dithiothreitol, MSL reacted with both the 40 and 43 kdalton proteins ($\text{6.0±0.6}\text{MSL}$ molecules per α-toxin-binding site). (3) After the latter labelling procedure membranes were exposed to pH 11, resulting in extraction of the 43 kdalton protein and leaving $\text{2.2 ± 0.4}\text{MSL}$ molecules per α-toxin-binding site; sodium dodecyl sulfate polyacrylamide gel electrophoresis performed with $\text{N-[}{}^{14}\text{C}\text{]}\text{ethylmaleimide}$ suggested that MSL was bound mainly to the 40 kdalton polypeptide chain of the acetylcholine receptor. The following conclusions were made with the native and alkaline-treated membranes: In the native membranes, saturation transfer ESR does not reveal any significant protein rotational diffusion (itrotational correlation time $\text{τ}{}_{\mathrm{c}}\text{> 1}\text{ms}$). Temperature variations and/or lipid modifications obtained by fusion of exogenous lipids and/or cholesterol exchange have little influence on the saturation transfer ESR spectra. Electron microscopy reveals that upon lipid addition, proteins remain in the form of clusters while areas depleted of proteins appear. On the other hand, alkaline treatment strikingly enhances the motion of the MSL-labelled proteins in the membrane ($\text{100 ? τ}{}_{\mathrm{c}}\text{? 120 μ}\text{s}$). Furthermore, the rotational diffusion of the MSL-labelled proteins (mainly the 40 kdalton protein) becomes sensitive to temperature, lipid composition and the lipid-to-protein ratio. Electron microscopy shows that alkaline extraction does not cause large reorganization of the acetylcholine receptor in the plane of the membrane. However, when phospholipids are added to pH 11 treated membranes, a dispersion of the receptor rosettes is observed. In contrast, cholesterol enrichment of the latter membranes induces clustering of the receptor and immobilization as judged by saturation transfer ESR. Upon reassociation of the pH 11 soluble proteins with the alkaline-treated membranes, the restriction of the acetylcholine receptor rotational mobility is also restored ($\text{τ}{}_{\mathrm{c}}\text{? 1}\text{ms}$).     

Vertical patterns and duration of surface wetness in an old-growth tropical montane forest, Indonesia

In tropical montane forests, the wetness of leaf surfaces is an important parameter which may influence gas exchange, growth and vitality of leaves, and forest productivity. Thirty surface wetness sensors were operated during May–August 2004 in a vertical profile inside an old-growth lower montane rain forest of Central Sulawesi, Indonesia, with the objective to analyse spatial and temporal patterns of surface wetness and to relate wetness duration to the microclimate inside the stand. The canopy was wet during 25–30% of time in this study period. In a dry period, however, surface wetness lasted for only 5% of the time, whereas the canopy was wet during 45–55% of the time in a rainy period. In the lower shade canopy, surface wetness continuously existed for periods of up to 22 h and more, although rainfall occurred only during afternoon thunderstorms of limited duration. The long duration of surface wetness has implications for forest interception models, which assume a complete drying of the canopy between subsequent rainfall events. In periods with rainfall, leaf wetness typically occurred in the afternoon, evening and first half of the night because intercepted water persisted on the leaves until about midnight. In dry periods, in contrast, surface wetness was mainly caused by dewfall in the second half of the night, and it occurred mainly in the uppermost canopy where radiative heat losses resulted in a substantial under-cooling of the leaves. Ecophysiological and hydrological importance is suggested by the long duration of surface wetting in this stand with possible implications for gas exchange, leaf growth, leaf colonization by epiphylls and the forest water balance.     

New horizons in culture and valorization of red microalgae

Research on marine microalgae has been abundantly published and patented these last years leading to the production and/or the characterization of some biomolecules such as pigments, proteins, enzymes, biofuels, polyunsaturated fatty acids, enzymes and hydrocolloids. This literature focusing on metabolic pathways, structural characterization of biomolecules, taxonomy, optimization of culture conditions, biorefinery and downstream process is often optimistic considering the valorization of these biocompounds. However, the accumulation of knowledge associated with the development of processes and technologies for biomass production and its treatment has sometimes led to success in the commercial arena. In the history of the microalgae market, red marine microalgae are well positioned particularly for applications in the field of high value pigment and hydrocolloid productions. This review aims to establish the state of the art of the diversity of red marine microalgae, the advances in characterization of their metabolites and the developments of bioprocesses to produce this biomass.     

Determination of the Monod substrate saturation constant for microbial growth

Abstract Methods used to determine the Monod substrate saturation constant for microbial growth are surveyed. The preferred and most accurate method is to assay the concentrations of growth rate-limiting nutrients in steady-state continuous cultures. But, this is not always possible due to the lack of sufficiently sensitive assay methods or due to high nutrient fluxes in rapidly growing cultures. It is suggested that an acceptable and simple alternative method for aerobic microorganisms is to measure initial oxygen uptake rates during growth in the presence of different initial concentrations of growth rate-limiting nutrient. It is important in this method that the microbial cells are taken from rapidly growing cultures and are suspended in a medium permitting growth.     

Application of 31P-NMR saturation transfer techniques to investigate phospholipid motion and organization in model and biological membranes

The potential of ³¹P-NMR saturation transfer experiments for determining motional characteristics (in the millisecond to second time scale) of phospholipids in model and biological membranes is demonstrated. A technique to separate membrane phospholipid ³¹P-NMR signals from those of small water-soluble phosphates in intact cells in liver tissue is also illustrated.     

Amino acid influx across the mucosal border of the rat intestine in vivo

The lack of an in vivo method for measuring influx of amino acid into the mucosa has prevented a systematic comparison of characteristics of amino acid influx in vivo with prior in vitro studies. We developed and validated a technique for measuring amino acid influx in vivo. The mucosa is exposed briefly to labelled amino acid perfused luminally at a rapid rate and tissue uptake is measured. The brief exposure period insures that amino acid is confined to the segment. The rapid perfusion rate minimizes concentration of endogenous Na⁺ in the lumen and permits Na-dependency for α-aminoisobutyric acid influx to be demonstrated in vivo for the first time. We also demonstrated the inhibitory effect of K⁺ and competition by glycine on α-aminoisobutyric acid influx in vivo. The saturation kinetics for l-leucine in vivo and in vitro were compared under varying perfusion rates and with and without stirring with air. Under optimal conditions of agitation (rapid perfusion and bubbling with air), the apparent Michaelis constant ($\text{K}{}_{\mathrm{<mtext>t</mtext>}}$) is decreased to be almost equal to that determined under comparable influx conditions in vitro. These studies demonstrate no major difference between characteristics of amino acid transport under more physiologic in vivo conditions as compared with prior in vitro studies.