Dual inhibition of monoamine oxidase B and antagonism of the adenosine A(2A) receptor by (E,E)-8-(4-phenylbutadien-1-yl)caffeine analogues

The adenosine A(2A) receptor has emerged as an attractive target for the treatment of Parkinson's disease (PD). Evidence suggests that antagonists of the A(2A) receptor (A(2A) antagonists) may be neuroprotective and may help to alleviate the symptoms of PD. We have reported recently that several members of the (E)-8-styrylcaffeine class of A(2A) antagonists also are potent inhibitors of monoamine oxidase B (MAO-B). Since MAO-B inhibitors are known to possess anti-parkinsonian properties, dual-target-directed drugs that block both MAO-B and A(2A) receptors may have enhanced value in the management of PD. In an attempt to explore this concept further we have prepared three additional classes of C-8 substituted caffeinyl analogues. The 8-phenyl- and 8-benzylcaffeinyl analogues exhibited relatively weak MAO-B inhibition potencies while selected (E,E)-8-(4-phenylbutadien-1-yl)caffeinyl analogues were found to be exceptionally potent reversible MAO-B inhibitors with enzyme-inhibitor dissociation constants (K(i) values) ranging from 17 to 149 nM. Furthermore, these (E,E)-8-(4-phenylbutadien-1-yl)caffeines acted as potent A(2A) antagonists with K(i) values ranging from 59 to 153 nM. We conclude that the (E,E)-8-(4-phenylbutadien-1-yl)caffeines are a promising candidate class of dual-acting compounds.     

Fungal virulence studies come of age

Sophisticated molecular biological research has revealed many virulence attributes in at least four pathogenic fungi, but the future study of fungal virulence requires investigators to distinguish between molecules that directly interact with the host, molecules that regulate these, and molecules that are always required for fungal growth and survival, independent of the host.     

1,3,4-Oxadiazol-2-ylbenzenesulfonamides as privileged structures for the inhibition of monoamine oxidase B

The present study investigates the monoamine oxidase (MAO) inhibition properties of a series of ten 5-aryl-1,3,4-oxadiazol-2-ylbenzenesulfonamides. The target compounds were synthesized by dehydration of the corresponding N,N′-diacylhydrazines with phosphorus oxychloride to yield the 1,3,4-oxadiazole cycle with concomitant transformation of the sulfonamide to the sulfonyl chloride group. Treatment with aqueous ammonia in acetonitrile regenerated the target sulfonamides. The results of the enzymology document that these compounds are potent and specific MAO-B inhibitors with the most potent compound exhibiting an IC₅₀ value of 0.0027 µM. An analysis of the structure-activity relationships shows that the 4-benzenesulfonamides are significantly more potent MAO-B inhibitors than the corresponding 3-benzenesulfonamides, and that the corresponding N,N′-diacylhydrazine synthetic precursors are weak MAO inhibitors. Although MAO inhibition by oxadiazole compounds are known, this is the first report of nanomolar MAO inhibition potencies recorded for sulfonamide derivatives. MAO-B specific inhibitors such as those discovered here may be of interest in the treatment of neurodegenerative disorders such as Parkinson’s disease.     

Examination of the Effects of Heterogeneous Organization of RyR Clusters,Myofibrils and Mitochondria on Ca2+ Release Patterns in Cardiomyocytes

Spatio-temporal dynamics of intracellular calcium, [Ca²⁺]_i, regulate the contractile function of cardiac muscle cells. Measuring [Ca²⁺]_i flux is central to the study of mechanisms that underlie both normal cardiac function and calcium-dependent etiologies in heart disease. However, current imaging techniques are limited in the spatial resolution to which changes in [Ca²⁺]_i can be detected. Using spatial point process statistics techniques we developed a novel method to simulate the spatial distribution of RyR clusters, which act as the major mediators of contractile Ca²⁺ release, upon a physiologically-realistic cellular landscape composed of tightly-packed mitochondria and myofibrils. We applied this method to computationally combine confocal-scale (~ 200 nm) data of RyR clusters with 3D electron microscopy data (~ 30 nm) of myofibrils and mitochondria, both collected from adult rat left ventricular myocytes. Using this hybrid-scale spatial model, we simulated reaction-diffusion of [Ca²⁺]_i during the rising phase of the transient (first 30 ms after initiation). At 30 ms, the average peak of the simulated [Ca²⁺]_i transient and of the simulated fluorescence intensity signal, F/F₀, reached values similar to that found in the literature ([Ca²⁺]_i ≈1 μM; F/F₀≈5.5). However, our model predicted the variation in [Ca²⁺]_i to be between 0.3 and 12.7 μM (~3 to 100 fold from resting value of 0.1 μM) and the corresponding F/F₀ signal ranging from 3 to 9.5. We demonstrate in this study that: (i) heterogeneities in the [Ca²⁺]_i transient are due not only to heterogeneous distribution and clustering of mitochondria; (ii) but also to heterogeneous local densities of RyR clusters. Further, we show that: (iii) these structure-induced heterogeneities in [Ca²⁺]_i can appear in line scan data. Finally, using our unique method for generating RyR cluster distributions, we demonstrate the robustness in the [Ca²⁺]_i transient to differences in RyR cluster distributions measured between rat and human cardiomyocytes.     

Integrating sequence and structural biology with DAS

Background  

The Distributed Annotation System (DAS) is a network protocol for exchanging biological data. It is frequently used to share annotations of genomes and protein sequence.     

Can Zipf's law be adapted to normalize microarrays?

Background  

Normalization is the process of removing non-biological sources of variation between array experiments. Recent investigations of data in gene expression databases for varying organisms and tissues have shown that the majority of expressed genes exhibit a power-law distribution with an exponent close to -1 (i.e. obey Zipf's law). Based on the observation that our single channel and two channel microarray data sets also followed a power-law distribution, we were motivated to develop a normalization method based on this law, and examine how it compares with existing published techniques. A computationally simple and intuitively appealing technique based on this observation is presented.     

Cellular determinants affecting the rate of cytokine in cultures of human hematopoietic cells

The present study was undertaken to define parameters that may limit the cytokine-mediated expansion of primitive hematopoietic cells in stirred suspension cultures of normal human marrow cells. In a first series of experiments, parallel measurements of the rate and extent of progenitor expansion and cytokine depletion from the medium were made for such cultures in which the cells were exposed to different cytokine concentrations. Supplementation of the medium with 2 ng/mL of interleukin-3 (IL-3), IL-6 and IL-11 plus 10 ng/mL of Flt-3 ligand (FL) and Steel factor (SF) allowed a 45-fold expansion of directly clonogenic cell (CFC) numbers within 2 weeks along with a 2.5-fold expansion of their precursors, detectable as longterm culture-initiating cells (LTC-IC). The addition of 5-fold higher levels of these cytokines enhanced the 2 week output of both CFC and LTC-IC numbers (to 66-fold and 9-fold above input respectively). However, this was also associated with an increase in the individual average rates of depletion of immunoreactive IL-3, SF and FL. As a result, even biweekly addition of fresh medium supplemented with the highest concentrations of cytokines tested failed to prevent a continuing decline in their levels relative to the input medium levels. A similar dependence of the IL-3 depletion rate on its extracellular concentration was demonstrable in suspension cultures of Mo7e cells, an IL-3-dependent human leukemic cell line.Additional experiments with various highly purified marrow cell fractions showed that the rate of cytokine depletion varied according to the type of responding cell as well as the specific cytokine. CD34(+)CD38(-) cells exhibited the greatest average cell-specific cytokine depletion rates (35-fold higher than unseparated bone marrow cells). These findings establish new principles that will be important for the optimization of hematopoietic cell bioreactors. In addition, they suggest that cytokine depletion may provide a novel feedback control mechanism in vivo which would contribute to the control of primitive hematopoietic cell proliferation and differentiation. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 58-66, 1997.