Kinetics of amyloid beta-protein degradation determined by novel fluorescence- and fluorescence polarization-based assays

Proteases that degrade the amyloid beta-protein (Abeta) are important regulators of brain Abeta levels in health and in Alzheimer's disease, yet few practical methods exist to study their detailed kinetics. Here, we describe robust and quantitative Abeta degradation assays based on the novel substrate, fluorescein-Abeta-(1-40)-Lys-biotin (FAbetaB). Liquid chromatography/mass spectrometric analysis shows that FAbetaB is hydrolyzed at closely similar sites as wild-type Abeta by neprilysin and insulin-degrading enzyme, the two most widely studied Abeta-degrading proteases. The derivatized peptide is an avid substrate and is suitable for use with biological samples and in high throughput compound screening. The assays we have developed are easily implemented and are particularly useful for the generation of quantitative kinetic data, as we demonstrate by determining the kinetic parameters of FAbetaB degradation by several Abeta-degrading proteases, including plasmin, which has not previously been characterized. The use of these assays should yield additional new insights into the biology of Abeta-degrading proteases and facilitate the identification of activators and inhibitors of such enzymes.     

Quantitative analysis of the fluorescence properties of intrinsically fluorescent proteins in living cells

The main potential of intrinsically fluorescent proteins (IFPs), as noninvasive and site-specific markers, lies in biological applications such as intracellular visualization and molecular genetics. However, photophysical studies of IFPs have been carried out mainly in aqueous solution. Here, we provide a comprehensive analysis of the intracellular environmental effects on the steady-state spectroscopy and excited-state dynamics of green (EGFP) and red (DsRed) fluorescent proteins, using both one- and two-photon excitation. EGFP and DsRed are expressed either in the cytoplasm of rat basophilic leukemia (RBL-2H3) mucosal mast cells or anchored (via LynB protein) to the inner leaflet of the plasma membrane. The fluorescence lifetimes (within approximately 10%) and spectra in live cells are basically the same as in aqueous solution, which indicate the absence of both IFP aggregation and cellular environmental effects on the protein folding under our experimental conditions. However, comparative time-resolved anisotropy measurements of EGFP reveal a cytoplasmic viscosity 2.5 +/- 0.3 times larger than that of aqueous solution at room temperature, and also provide some insights into the LynB-EGFP structure and the heterogeneity of the cytoplasmic viscosity. Further, the oligomer configuration and internal depolarization of DsRed, previously observed in solution, persists upon expression in these cells. DsRed also undergoes an instantaneous three-photon induced color change under 740-nm excitation, with efficiently nonradiative green species. These results confirm the implicit assumption that in vitro fluorescence properties of IFPs are essentially valid for in vivo applications, presumably due to the beta-barrel protection of the embodied chromophore. We also discuss the relevance of LynB-EGFP anisotropy for specialized domains studies in plasma membranes.     

Analgesic activity of Piper longum Linn. root

Piper longum root, commonly called Kandantippili, is traditionally used to treat rheumatism, insomnia, palsy and epilepsy. But a scientific study on its central actions is not available. This study screens P. longum root for opioid type analgesia using rat tail-flick method and for NSAID type analgesia using acetic-acid writhing method. Pentazocine (ip) and ibuprofen (oral) are used as respective drug controls. An aqueous suspension of P. longum root powder is given orally to mice and rat in doses of 200, 400 and 800 mg/kg. The delay in reaction time for thermal stimulus in rats and the number of writhings to chemical stimulus in mice are determined in each group. The results are analysed statistically. The 400 and 800 mg/kg doses of P. longum show significant NSAID type of analgesia (P < 0.001). Both Ibuprofen (40 mg/kg) and P. longum (800 mg/kg) show 50% protection against writhing. The delay in reaction time to thermal stimulus was less than 6% for different doses of P. longum as against 100% for pentazocine. This indicates that P. longum root has weak opioid but potent NSAID type of analgesic activity.     

In vitro and in vivo evaluations of biodegradable implants for hormone replacement therapy: Effect of system design and PK-PD relationship

This investigation evaluated the feasibility of using subdermally implantable devices fabricated by nonconventional 3-dimensional printing technology for controlled delivery of ethinyl estradiol (EE₂). In vitro release kinetics of EE₂ and in vivo pharmacokinetics pharmacodynamics in ovariectomized New Zealand White rabbits were carried out to study 3 implant prototypes: implant I (single-channel EE₂ distribution in polycaprolactone polymer core), implant II (homogeneous EE₂ distribution in polycaprolactone polymer matrix), and implant III (concentration-gradient EE₂ distribution in polycaprolactone and poly(dl-lactide-co-glycolide) (50∶50 matrix). EE₂ was found to be released from all the implants in a nonlinear pattern with an order of implant III>implant II>implant I. The noncompartmental pharmacokinetic analysis of plasma EE₂ profiles in rabbits indicated a significant difference (p>.05) in C_max, t_max, and mean residence time between implant I and implants II and III, but no difference in the area under the plasma concentration time curves calculated by trapezoidal rule (AUC) among the implants. For pharmacodynamic studies, endogenous follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels were observed to be suppressed following implantation of all implants, which demonstrated that a therapeutically effective dose of EE₂ had been delivered. Furthermore, the noncompartmental analysis of plasma FSH and LH profiles in rabbits showed a significant difference (p<.05) in AUC and the mean residence time between implant III and implants I and II. A good in vivo/in vitro relationship was observed between daily amounts of EE₂ released and plasma profiles of EE₂ for all implants. This relationship suggests that plasma profiles of EE₂ could be predicted from in vitro measurement of daily amount of EE₂ released Therefore, performing in vitro drug release studies may aid in the development of an EE₂ implant with the desired in vivo release rate. Published: September 21, 2001     

Transepithelial organic anion transport by shark choroid plexus

Spiny dogfish shark (Squalus acanthias) lateral and IV choroid plexuses (CPs) are ultrastructurally similar to the corresponding tissues of rat. However, shark IV CP is proportionally larger and easily accessible. Moreover, this epithelial sheet can be halved and studied in Ussing flux chambers. We have used confocal fluorescence microscopy and radiotracer techniques to characterize transepithelial transport of the organic anions (OAs) fluorescein (FL) and 2,4-dichlorophenoxyacetic acid (2,4-D), respectively, by shark CP. Lateral and IV CP accumulated 1 microM FL, with highest levels in the underlying extracellular spaces, intermediate levels in epithelial cells, and lowest levels in the medium. 2,4-D and probenecid inhibited FL accumulation in cells and extracellular spaces, suggesting that these substrates compete for common carriers. Unidirectional absorptive [cerebrospinal fluid (CSF)-to-blood] and secretory (blood-to-CSF) fluxes of 10 microM [(14)C]2,4-D were measured under short-circuited conditions in IV CP mounted in Ussing chambers. 2,4-D underwent net absorption, with an average flux ratio of 7. Probenecid, 2,4,5-trichlorophenoxyacetic acid, and 5-hydroxyindolacetic acid reduced net absorption, reversibly inhibiting unidirectional absorption, with no effect on secretion. Ouabain irreversibly reduced net 2,4-D absorption and cellular and extracellular accumulation of FL, suggesting energetic coupling of OA absorption to Na(+) transport. Collectively, these data indicate that shark CP actively removes OAs from CSF by a process that is specific and active.     

Premature chromosome condensation in humans associated with microcephaly and mental retardation: a novel autosomal recessive condition

We report a novel autosomal recessive disorder characterized by premature chromosome condensation in the early G2 phase. It was observed in two siblings, from consanguineous parents, affected with microcephaly, growth retardation, and severe mental retardation. Chromosome analysis showed a high frequency of prophase-like cells (>10%) in lymphocytes, fibroblasts, and lymphoblast cell lines with an otherwise normal karyotype. (3)H-thymidine-pulse labeling and autoradiography showed that, 2 h after the pulse, 28%-35% of the prophases were labeled, compared with 9%-11% in healthy control subjects, indicating that the phenomenon is due to premature chromosome condensation. Flow cytometry studies demonstrate that the entire cell cycle is not prolonged, compared with that in healthy control subjects, and compartment sizes did not differ from those in healthy control subjects. No increased reaction of the cells to X-irradiation or treatments with the clastogens bleomycin and mitomycin C was observed, in contrast to results in the cell-cycle mutants ataxia telangiectasia and Fanconi anemia. The rates of sister chromatid exchanges and the mitotic nondisjunction rates were inconspicuous. Premature entry of cells into mitosis suggests that a gene involved in cell-cycle regulation is mutated in these siblings.     

Morphology and morphometry of in vivo- and in vitro-produced bovine concepti from early pregnancy to term and association with high birth weights

This study was designed to characterize conceptus development based on pre- and postnatal measurements of in vivo- and in vitro-derived bovine pregnancies. In vivo-produced embryos were obtained after superovulation, whereas in vitro-produced embryos were derived from established procedures for bovine IVM, IVF and IVC. Blastocysts were transferred to recipients to obtain pregnancies of single (in vivo/singleton or in vitro/singleton groups) or twin fetuses (in vitro/twins group). Ultrasonographic examinations were performed weekly, from Day 30 of gestation through term. Videotaped images were digitized, and still-frames were used for the measurement of conceptus traits. Calves and fetal membranes (FM) were examined and measured upon delivery. In vitro-produced fetuses were smaller than in vivo controls (P < 0.05) during early pregnancy (Day 37 to Day 58), but in vitro/singletons presented significantly higher weights at birth than in vivo/control and in vitro/twin calves (P < 0.05). From late first trimester of pregnancy (Day 72 to Day 93), placentomes surrounding in vitro-derived singleton fetuses were longer and thinner than controls (P < 0.05). At term, the presence of giant cotyledons in the fetal membranes in the in vitro group was associated with a larger cotyledonary surface area in the fetal horn (P < 0.05). The biphasic growth pattern seen in in vitro-produced pregnancies was characterized by conceptus growth retardation during early pregnancy, followed by changes in the development of the placental tissue. Resulting high birth weights may be a consequence of aberrant placental development due to the disruption of the placental restraint on fetal growth toward the end of pregnancy.     

Effects of dietary fat source and subtherapeutic levels of antibiotic on the bacterial community in the ileum of broiler chickens at various ages

The effect of dietary fat source (soy oil or a mixture of lard and tallow) and dietary supplementation with antibiotics (a combination of avilamycin at 10 mg kg of feed(-1) and salinomycin at 40 mg kg of feed(-1)) on the bacterial community in the ileum of broiler chickens at different ages (7, 14, 21, and 35 days) was studied using PCR with denaturing gradient gel electrophoresis (DGGE) analysis and bacteriological culture. The bacterial origin of fragments in DGGE profiles was identified by sequencing. Bacterial enumeration results, together with PCR-DGGE profiles, showed that the composition of the microflora was age dependent and influenced by dietary fat source and antibiotic supplementation. An increased incidence of streptococci, enterobacteria, and Clostridium perfringens with age of the chickens was demonstrated. Lactobacilli and C. perfringens were the bacterial groups most strongly affected by the dietary treatments. Moreover, different strains (clonal variants of the alpha-toxin gene) of C. perfringens type A were detected in response to age, dietary fat source, and dietary supplementation with antibiotics.