MS and NMR investigation of bioactive quinazolones

To prepare effective PARP [poly(ADP)-ribose)polymerase-1] inhibitors, starting from 2-mercapto-4(3H)-quinazolone (1), several S-alkyl derivatives--2-alkylsulfanyl-3H-quinazolin-4-ones (2-5, 7-9)--as well as an S-benzyl derivative (10) were prepared using a simple alkylation method. Some of them are known compounds. Their structure was studied thoroughly by MS and NMR methods.     

Presynaptic alpha2-receptors regulate reverse Na+/Ca2+-exchange and transmitter release in Na+-loaded peripheral sympathetic nerves

Electrical depolarisation-(2 Hz, 1 ms)-induced [3H]noradrenaline ([3H]NA) release has been measured from the isolated main pulmonary artery of the rabbit in the presence of uptake blockers (cocaine, 3 x 10(-5) M; corticosterone, 5 x 10(-5) M). Substitution of most of the external Na+ by Li+ (113 mM; [Na+]0: 25 mM) slightly potentiated the axonal stimulation-evoked release of [3H]NA in a tetrodotoxin (TTX, 10(-7) M) sensitive manner. The reverse Na+/Ca2+-exchange inhibitor KB-R7943 (3 x 10(-5) M) failed to inhibit the stimulation-evoked release of [3H]NA, but increased the resting outflow of neurotransmitter. The 'N-type' voltage-sensitive Ca2+-channel (VSCC) blocker omega-conotoxin (omega-CgTx) GVIA (10(-8) M) significantly and irreversibly inhibited the release of [3H]NA on stimulation (approximately 60-70%). The 'residual release' of NA was abolished either by TTX or by reducing external Ca2+ from 2.5 to 0.25 mM. The 'residual release' of NA was also blocked by the non-selective VSCC-blocker neomycin (3 x 10(-3) M). Correlation was obtained between the extent of VSCC-inhibition and the transmitter release-enhancing effect of presynaptic alpha2-receptor blocker yohimbine (3 x 10(-7) M). When the release of [3H]NA was blocked by omega-CgTx GVIA plus neomycin, yohimbine was ineffective. Inhibition of the Na+-pump by removal of K+ from the external medium increased both the resting and the axonal stimulation-evoked release of [3H]NA in the absence of functioning VSCCs (i.e., in the presence of neomycin and after omega-CgTx treatment). Under these conditions the stimulation-evoked release of NA was abolished either by TTX or by external Ca2+-removal (+1 mM EGTA). Similarly, external Li+ (113 mM) or the reverse Na+/Ca2+ exchange blocker KB-R7943 (3 x 10(-5) M) significantly inhibited the stimulation-induced transmitter release in 'K+-free' solution. KB-R7943 decreased the resting outflow of NA as well. Under conditions in which the Na+-pump was inhibited in the absence of functioning VSCCs, yohimbine (3 x 10(-7) M) further enhanced the release of neurotransmitter, while l-noradrenaline (l-NA, 10(-6) M), an agonist of presynaptic alpha2-receptors, inhibited it. The yohimbine-induced enhancement of NA-release was abolished by Li+-substitution and significantly inhibited by KB-R7943 application. It is concluded that after blockade of VSCCs brief depolarising pulses may reverse Na+/Ca2+-exchange and release neurotransmitter in Na+-loaded sympathetic nerves. Further, similar to that of VSCCs, the reverse Na+/Ca2+-exchange may also be regulated by presynaptic alpha2-receptors.     

Dendritic cells: sentinels against pathogens

Dendritic cells (DCs) are the most potent antigen-presenting cells, and are regarded as "natural adjuvants" for the induction of primary T or T-dependent immunity. DCs in the peripheral sites capture and process antigens. Encounter of exogenous or endogenous stimuli mature the function of DCs, and they thus acquire T-cell stimulatory capacity and distinct chemotactic behavior which enables them to migrate to lymphoid tissue. In the secondary lymphoid organs, they present antigens to T- and B-cells and stimulate their proliferation. Dendritic cells are also involved in tolerance induction, in particular, to self antigens. DCs also play a key role in the transmission of many pathogens, and therefore may become targets for designing new therapies. DCs have been manipulated in vitro and in vivo for cancer immunotherapy. In this article, we provide a concise overview of DC biology and its current and future role in clinical settings.     

Discovery of novel aspartyl ketone dipeptides as potent and selective caspase-3 inhibitors

The discovery of a series of potent, selective and reversible dipeptidyl caspase-3 inhibitors are reported. The iterative discovery process of using combinatorial chemistry, parallel synthesis, moleculare modelling and structural biology will be discussed.     

Extraction of fluorescent cell puncta by adaptive fuzzy segmentation

MOTIVATION: The discrimination and measurement of fluorescent-labeled vesicles using microscopic analysis of fixed cells presents a challenge for biologists interested in quantifying the abundance, size and distribution of such vesicles in normal and abnormal cellular situations. In the specific application reported here, we were interested in quantifying changes to the population of a major organelle, the peroxisome, in cells from normal control patients and from patients with a defect in peroxisome biogenesis. In the latter, peroxisomes are present as larger vesicular structures with a more restricted cytoplasmic distribution. Existing image processing methods for extracting fluorescent cell puncta do not provide useful results and therefore, there is a need to develop some new approaches for dealing with such a task effectively. RESULTS: We present an effective implementation of the fuzzy c-means algorithm for extracting puncta (spots), representing fluorescent-labeled peroxisomes, which are subject to low contrast. We make use of the quadtree partition to enhance the fuzzy c-means based segmentation and to disregard regions which contain no target objects (peroxisomes) in order to minimize considerable time taken by the iterative process of the fuzzy c-means algorithm. We finally isolate touching peroxisomes by an aspect-ratio criterion. The proposed approach has been applied to extract peroxisomes contained in several sets of color images and the results are superior to those obtained from a number of standard techniques for spot extraction. AVAILABILITY: Image data and computer codes written in Matlab are available upon request from the first author.     

Genotyping over 100,000 SNPs on a pair of oligonucleotide arrays

We present a genotyping method for simultaneously scoring 116,204 SNPs using oligonucleotide arrays. At call rates >99%, reproducibility is >99.97% and accuracy, as measured by inheritance in trios and concordance with the HapMap Project, is >99.7%. Average intermarker distance is 23.6 kb, and 92% of the genome is within 100 kb of a SNP marker. Average heterozygosity is 0.30, with 105,511 SNPs having minor allele frequencies >5%.     

Activated platelets rapidly up-regulate CD40L expression and can effectively mature and activate autologous ex vivo differentiated DC

Background DC are a promising immunotherapeutic for treatment of infectious/malignant disease. For clinical trials, immature DC generated from precursor cells such as monocytes, using serum-free media containing GM-CSF and IL-4, can be matured with specific cytokines/factors. CD40 ligand (CD40L) plays an important role in DC activation/maturation but is not available for clinical applications. These studies evaluated the feasibility of using activated platelets with elevated CD40L surface expression to stimulate autologous DC maturation. Methods Pilot and clinical scale studies were executed using magnetic/centrifugal separation. Monocyte precursors were differentiated to immature DC with GM-CSF and IL-4 and the ability of activated autologous platelets to mature these cells was evaluated on the basis of phenotype and function. Results In small-scale studies, DC cultures stimulated with activated autologous platelets (CD40L-AP), tumor necrosis factor-alpha (TNF-alpha) or soluble CD40L (sCD40L) up-regulated expression of phenotype markers indicative of activation and maturation. CD86 expression was significantly enhanced (P<0.05) by stimulation with either CD40L-AP (75.5+/-14.5%) or sCD40L (80.5%+/-5.3%) compared with immature DC (55.2+/-14.8%), as were CD80 and CD83. Similarly, in large-scale studies using Isolex 300I to enrich for monocytes and platelets for DC generation/maturation on a clinical scale, stimulation with CD40L-AP increased CD86 and CD80 expression as well as the ability to stimulate allogeneic lymphocytes compared with control cultures. Discussion These results demonstrate that thrombin-activated platelets express CD40L and are effective at inducing matured DC with potent immunogenic activity. Furthermore, these studies demonstrate the feasibility of this approach for clinical immunotherapeutic interventions.     

Depletion and fractionation technologies in plasma proteomic analysis

This review intends to survey the traditional and current technologies in the depletion and subfractionation of plasma proteins for further analyses. The value of depletion aims to enrich low-abundant proteins by removing highly abundant proteins, such as albumin or immunoglobulin G, from plasma. With this approach, one can examine both the resulting high- and low-abundant protein fractions. The depleted protein population can be further subfractionated based on their isoelectric point ranges, creating a more discrete pool of proteins for detailed post-translational modification studies by methods such as 2D gel electrophoresis and mass spectrometry. The concept of divide to conquer will greatly enhance our ability to identify and characterize low-abundant proteins and cleaved peptides from plasma as important diagnostic markers or potential drug targets. This can potentially reverse the decline in the development of new plasma diagnostic tests.     

Embryonic axes: the long and short of it in the mouse

Contrary to conventional wisdom, recent studies indicate that, during mouse development, the embryo's anterior-posterior axis shifts from the shorter transverse axis to the orthogonal longer one as the shape of the pre-gastrula-stage embryo is remodelled.     

Nocardia asteroides culture filtrates cause dopamine depletion and cytotoxicity in PC12 cells

Experimental infection of BALB/c mice with the gram-positive bacterium Nocardia asteroides produces marked loss of nigrostriatal dopamine neurons, resulting in striatal dopamine depletion. To investigate the mechanism(s) responsible for this neuronal loss, we evaluated the influence of N. asteroides cell-free culture filtrates on rat pheochromocytoma PC12 cells, an in vitro model for dopamine neurons. Changes in cell viability and cell numbers were minimal after 24 h, but increased with longer incubation. In contrast, dopamine depletion occurred after 30 min incubation, and was greater with GUH-2 filtrate than with filtrate from the less virulent strain 10905. Incubation with the culture filtrate decreased viability in neuroblastoma and glioma cell lines, indicating that cytotoxic effects were not limited to dopaminergic cells. These findings suggest that the loss of nigrostriatal dopamine neurons and concomitant striatal dopamine depletion in Nocardia-infected mice may be due, at least in part, to the neurotoxicity of nocardial secretory products.