Long-Term Safety of Repeated Blood-Brain Barrier Opening via Focused Ultrasound with Microbubbles in Non-Human Primates Performing a Cognitive Task

Focused Ultrasound (FUS) coupled with intravenous administration of microbubbles (MB) is a non-invasive technique that has been shown to reliably open (increase the permeability of) the blood-brain barrier (BBB) in multiple in vivo models including non-human primates (NHP). This procedure has shown promise for clinical and basic science applications, yet the safety and potential neurological effects of long term application in NHP requires further investigation under parameters shown to be efficacious in that species (500kHz, 200–400 kPa, 4–5μm MB, 2 minute sonication). In this study, we repeatedly opened the BBB in the caudate and putamen regions of the basal ganglia of 4 NHP using FUS with systemically-administered MB over 4–20 months. We assessed the safety of the FUS with MB procedure using MRI to detect edema or hemorrhaging in the brain. Contrast enhanced T1-weighted MRI sequences showed a 98% success rate for openings in the targeted regions. T2-weighted and SWI sequences indicated a lack edema in the majority of the cases. We investigated potential neurological effects of the FUS with MB procedure through quantitative cognitive testing of’ visual, cognitive, motivational, and motor function using a random dot motion task with reward magnitude bias presented on a touchpanel display. Reaction times during the task significantly increased on the day of the FUS with MB procedure. This increase returned to baseline within 4–5 days after the procedure. Visual motion discrimination thresholds were unaffected. Our results indicate FUS with MB can be a safe method for repeated opening of the BBB at the basal ganglia in NHP for up to 20 months without any long-term negative physiological or neurological effects with the parameters used.     

Ultrastructural modifications of the cavities formed by folliculo-stellate cells in chicken adenohypophysis under septic shock conditions

Effects of septic shock by repeated inoculations with Escherichia coli on the ultrastructure of the folliculo-stellate cells and cavities of the adenohypophysis of the chicken were investigated in order to determine the function of these cavities. The principal morphological modifications were dilation of the Golgi apparatus, endoplasmic reticulum and autophagic vacuoles, and necrosis phenomena in the stellate cells. The follicular cavities showed dilation, and there was heterogeneous dense material and granular elements in the follicular lumen. Based on results reported in the literature, the observations reported here are evidence of a "cleaning-role", for the removal of cell debris, when there is endocrine disfunction.     

A microassay for heme oxygenase activity using thin-layer chromatography.

A sensitive and facile assay for heme oxygenase (HO) has been developed. The basis of the assay is the detection of [14C]bilirubin formation in a coupled enzyme assay involving HO and biliverdin reductase actions, respectively. Separation of substrate from product is accomplished by thin-layer chromatography with subsequent quantitation by liquid scintillation counting of radioactive material present on chromatograms. As little as 20 micrograms of total cellular protein derived from cells growing in a standard 25-cm2 culture flask is sufficient for detection of HO enzyme activity using this assay. The reaction is inhibited by tin-protoporphyrin (10 microM final concentration), a specific inhibitor of HO. The linearity of the enzyme reaction with respect to incubation time and amount of protein used was established. Comparison of the new HO assay with a spectrophotometric assay was made, and good agreement of the results from both methods was found. The assay described here should facilitate measurements of this important heme-degrading enzyme in tissue culture studies and cases where limited amounts of material are available.     

Staphylococcal growth and enterotoxins (A—D) and thermonuclease synthesis in the presence of dehydrated garlic

E. GONZÁLEZ-FANDOS, M.L. GARCÍA-LÓPEZ, M.L. SIERRA AND A. OTERO. 1994. The inhibition of Staphylococcus aureus growth and enterotoxin and thermonuclease production by various concentrations of garlic ( Allium sativum ) was studied in BHI broth. The growth of Staph. aureus was inhibited by dehydrated garlic at levels of 1.5% (w/v) and over. Enterotoxins A, B and C₁ were only detectable in broth containing < 1% of garlic while enterotoxin D was produced at a level of 2%. Garlic also inhibited thermonuclease (TNAse) production, complete inhibition being observed at levels ≥ 1.5%. TNAse was not always detected when enterotoxin was present.     

Synthesis and biological evaluation of novel NK-1 tachykinin receptor antagonists: The use of cycloalkyl amino acids as a template

In the course of a program aimed at synthesizing novel, potent NK-1 tachykinin receptor antagonists, we developed upon a bioactive model by comparing the low energy structures of a series of peptide and nonpeptide Substance P antagonists. The comparison was based on the super imposition of the aromatic rings, assuming that the rest of the molecule behaves predominantly as a template to arrange the key aromatic groups in the right spatial position. A series of 2-aminocyclohexane carboxylic acid analogues were then selected as the best templates for reproducing the postulated bioactive structure, leading to several pseudo-peptides with interesting biological activity. According to the molecular modeling, these compounds exhibit a neat parallel facing of the indolyl and naphthyl groups at about 3 Å distance. Ultraviolet absorption and steady state fluorescence measurements support this conclusion, showing a linear correlation between the spectral properties and the binding affinity of these analogues. Stacking of the indole ring with naphthalene gives rise to a complex characterized by a well-defined molar extinction coefficient. Consistently, steady state and lifetime fluorescence measurements suggest that the quenching process is ascribable to ground-state interactions between the chromophores. Implications of the π stacking propensity of aromatic groups in the biological activity of the compounds examined are briefly discussed. © 1995 John Wiley & Sons, Inc.     

Time-resolved fluorescence study of human recombinant interferon alpha 2. Association state of the protein, spatial proximity of the two tryptophan residues.

Human recombinant interferon alpha 2 belongs a to family of proteins active against a wide range of viruses. It contains two tryptophan residues located at positions 77 and 141 in the peptide sequence. The fluorescence emission spectrum of these tryptophan residues displays a maximum at 335 nm. The fluorescence intensity decay is described by one broad excited-state-lifetime population centered around a value of 1.7 ns (full width at half maximum, 1.5 ns). These observations suggest that in the native protein, both tryptophan residues emit from similar environments, not directly exposed to the surrounding solvent. The anisotropy decay is essentially biexponential. The correlation-time value characterizing the Brownian rotation of the protein varies linearly with the viscosity/temperature ratio. The calculated hydrodynamic volumes are compatible with the existence of a dimer and a tetramer, at pH 5.5 and 9.4, respectively. Addition of urea at pH 5.5 disrupts the dimer and modifies to some extent the excited-state-lifetime distribution which becomes more heterogeneous. Disulfide-bond reduction also dissociates the dimer and leads to a highly heterogeneous fluorescence-intensity decay with four excited-state-lifetime populations. An opening of the local structure in the Trp region of the protein is likely to occur in these conditions. The fast-anisotropy-decay components can be due to either fast rotation or energy transfer between the indoles. Close proximity of the two Trp residues (less than 1 nm) is suggested from steady-state and time-resolved fluorescence-anisotropy measurements in vitrified medium [95% (by mass) glycerol at -38 degrees C]. This suggestion is in agreement with the recently published three-dimensional structure of the homologous protein murine interferon beta [Senda, T., Shimazu, T., Matsuda, S. Kawano, G., Shimizu, H., Nakamura, K. T. & Mitsui, Y. (1992) EMBO J. 11, 3193-3201].