Novel amidino substituted 2-phenylbenzothiazoles: Synthesis,antitumor evaluation in vitro and acute toxicity testing in vivo

The efficient synthesis of new bis-substituted nitro-amidino, amino-amidino (10a, 10b–13a, 13b) and previously prepared diamidino 2-phenyl-benzothiazoles (9a, 9b) is described. The compounds 11a and 11b were prepared by recently developed methodology of the key precursors in zwitterionic form 8a and 8b with 4-nitrobenzoylchloride in a very good yield (70%). All compounds except diamidino-substituted 2-phenylbenzothiazole 9a show exceptionally prominent tumor cell-growth inhibitory activity and cytotoxicity, whereby the special selectivity of amino-amidine 2-phenylbenzothiazole 12a towards MCF-7 and H 460 cells makes this compound a prospective lead compound that should be further evaluated in animal models. All in vivo tested compounds (12a, 12b, 13a and 13b) are absorbed from mice gastrointestinal system. LD₅₀ are between 67.33 and 696.2 mg/kg body weight (OECD/EPA toxicity categories 2–3).     

Immobilization of lipase from Candida rugosa on Eupergit C supports by covalent attachment

The present study compares the results of three different covalent immobilization methods employed for immobilization of lipase from Candida rugosa on Eupergit^® C supports with respect to enzyme loadings, activities and coupling yields. It seems that method yielding the highest activity retention of 43.3% is based on coupling lipase via its carbohydrate moiety previously modified by periodate oxidation. Study of thermal deactivation kinetics at three temperatures (37, 50 and 75 °C) revealed that the immobilization method also produces an appreciable stabilization of the biocatalyst, changing its thermal deactivation profile. By comparison of the t_1/2 values obtained at 75 °C, it can be concluded that the lipase immobilized via carbohydrate moiety was almost 2-fold more stable than conventionally immobilized one and 18-fold than free lipase. The immobilization procedure developed is quite simple, and easily reproduced, and provides a promising solution for application of lipase in aqueous and microaqueous reaction system.     

Lightness, illumination, and gradients

The illumination interpretation approach claims that lightness illusions can be explained as misapplications of lightness constancy mechanisms, processes which usually enable veridical extraction of surface reflectance from luminance distributions by discounting illumination. In particular, luminance gradients are thought to provide cues about the interactions of light and surfaces. Several examples of strong lightness illusions are discussed for which explanations based on illumination interpretation can be proposed. In criticisms of this approach, a variety of demonstrations of similarly structured control displays are presented, which involve equivalent lightness effects that cannot readily be accounted for by illumination interpretation mechanisms. Furthermore, a number of known and novel displays are presented that demonstrate effects of gradients on the qualitative appearance of uniform regions. Finally, some simple simulations of neural effects of luminance distributions are discussed.     

Estimating Lymphocyte Division and Death Rates from CFSE Data

The division tracking dye, carboxyfluorescin diacetate succinimidyl ester (CFSE) is currently the most informative labeling technique for characterizing the division history of cells in the immune system. Gett and Hodgkin [Nat. Immunol. 1:239–244, 2000] have pioneered the quantitative analysis of CFSE data. We confirm and extend their data analysis approach using simple mathematical models. We employ the extended Gett and Hodgkin [Nat. Immunol. 1:239–244, 2000] method to estimate the time to first division, the fraction of cells recruited into division, the cell cycle time, and the average death rate from CFSE data on T cells stimulated under different concentrations of IL-2. The same data is also fitted with a simple mathematical model that we derived by reformulating the numerical model of Deenick et al. [J. Immunol. 170:4963–4972, 2003]. By a non-linear fitting procedure we estimate parameter values and confidence intervals to identify the parameters that are influenced by the IL-2 concentration. We obtain a significantly better fit to the data when we assume that the T cell death rate depends on the number of divisions cells have completed. We provide an outlook on future work that involves extending the Deenick et al. [J. Immunol. 170:4963–4972, 2003] model into the classical smith-martin model, and into a model with arbitrary probability distributions for death and division through subsequent divisions.     

Analysis of cycloheximide-induced apoptosis in human leukocytes: fluorescence microscopy using annexin V/propidium iodide versus acridin orange/ethidium bromide

Apoptosis is a highly regulated and programmed cell breakdown process characterized by numerous changes. Since it is implicated in many pathological as well as physiological processes, it is vital to have reliable methods for detecting cell death. In this study, we compared several methods for detecting apoptosis and necrosis in human leukocytes. Apoptosis was induced either by incubating the cells with various doses of cycloheximide (CHX) or by 312 nm UVB irradiation. The methods used for detecting apoptosis were light microscopy (May Grunwald-Giemsa and trypan blue staining), fluorescence microscopy (acridin orange/ethidium bromide and annexin V/propidium iodide staining) and agarose gel electrophoresis of fragmented genomic DNA. Our study showed that CHX-induced apoptosis in cultured peripheral blood mononuclear cells but had no effect on apoptosis in polymorphonuclear cells, so its effect depends on cell type. Evaluation and comparison of the methods for detecting apoptosis showed the following. A Giemsa-stained cytospin allows the main morphological characteristics of necrotic and apoptotic death to be recognized. Trypan blue staining, widely used for estimating cell viability, is valueless for detecting apoptosis. Both fluorescence methods provided reliable and reproducible results and distinguished clearly between subpopulations of apoptotic cells, and were closely intercorrelated. Although applicable to a wide spectrum of cell types, agar electrophoresis of extracted DNA cannot be applied to all cell types and apoptotic conditions. Generally, microscopic examination of acridin orange/ethidium bromide stained cells can be recommended as the most reliable of the methods tested.     

Monitoring lysin motif-ligand interactions via tryptophan analog fluorescence spectroscopy

The lysin motif (LysM) is a peptidoglycan binding protein domain found in a wide range of prokaryotes and eukaryotes. Various techniques have been used to study the LysM-ligand interaction, but a sensitive spectroscopic method to directly monitor this interaction has not been reported. Here a tryptophan analog fluorescence spectroscopy approach is presented to monitor the LysM-ligand interaction using the LysM of the N-acetylglucosaminidase enzyme of Lactococcus lactis. A three-dimensional model of this LysM protein was built based on available structural information of a homolog. This model allowed choosing the amino acid positions to be labeled with a Trp analog. Four functional single-Trp LysM mutants and one double-Trp LysM mutant were constructed and biosynthetically labeled with 7-azatryptophan or 5-hydroxytryptophan. These Trp analogs feature red-shifted absorption spectra, enabling the monitoring of the LysM-ligand interaction in media with a Trp background. The emission intensities of four of the five LysM constructs were found to change markedly on exposure to either L. lactis bacterium-like particles or peptidoglycan as ligands. The method reported here is suitable to monitor LysM-ligand interactions at (sub)micromolar LysM concentrations and can be used for the detection of low levels of peptidoglycan or microbes in solutions.     

High Diversity of Indigenous Populations of Dalmatian Sage (Salvia officinalis L.) in Essential‐Oil Composition

Essential oils of 25 indigenous populations of Dalmatian sage (Salvia officinalis L.) that represent nearly half of native distribution area of the species were analyzed. Plantlets collected from wild populations were grown in the same field under the same environmental conditions and then sampled for essential‐oil analysis. The yield of essential oil ranged from 1.93 to 3.70% with average of 2.83%. Among the 62 compounds detected, eight (cis‐thujone, camphor, trans‐thujone, 1,8‐cineole, β‐pinene, camphene, borneol, and bornyl acetate) formed 78.13–87.33% of essential oils of individual populations. Strong positive correlations were observed between camphor and β‐pinene, β‐pinene and borneol, as well as between borneol and bornyl acetate. The strongest negative correlation was detected between camphor and trans‐thujone. Principal component analysis (PCA) on the basis of eight main compounds showed that first main component separated populations with high thujone content, from those rich in camphor, while the second component separated populations rich in cis‐thujone from those rich in trans‐thujone. Cluster analysis (CA) led to the identification of three chemotypes of S. officinalis populations: cis‐thujone; trans‐tujone, and camphor/β‐pinene/borneol/bornyl acetate. We propose that differences in essential oils of 25 populations are mostly genetically controlled, since potential environmental factors were controlled in this study.     

Adaptation of the gypsy moth to an unsuitable host plant

The pattern of adaptation with regard to life history traits and traits thought to be important in feeding habits of caterpillars in two populations of the gypsy moth (Lymantria dispar L.; Lepidoptera: Lymantriidae) originating from the locust tree (Robinia pseudoacacia; Fabaceae) and oak (Quercus petrea; Fagaceae) forests were investigated in the laboratory. The Robinia population has experienced unsuitable locust tree leaves as an exclusive food resource for more than 40 years. Since Quercus species are the principal host plants of the gypsy moth, the specific objectives of this study have been to measure the extent of differentiation between ancestral and derived populations in several life history traits (egg-to-adult viability, duration of larval and pupal stages, and pupal weight) and nutritional indices – relative growth rate (RGR), relative consumption rate (RCR), assimilation efficiency (AD), gross growth efficiency (ECI), and net growth efficiency (ECD). Significant differences between the Quercus and Robinia populations were detected in pupal duration, RGR, RCR, and AD. The presence of a significant population × host interaction in traits such as preadult viability, duration of pupal stage, RGR, and ECI suggests that adaptation of the gypsy moth to the unsuitable host might be ongoing. Using a full-sib design, we screened for genetic variation in life history traits within both populations, and examined the genetic correlations of performance across oak and locust leaves within both populations. The genetic variances for analyzed life history traits were lower under conditions that are commonly encountered in nature. Our data show that positive cross-host genetic correlations preponderate within both populations.     

Liposome-entrapped superoxide dismutase reduces ischemia/reperfusion ‘oxidative stress’ in gerbil brain

Bilateral common carotid artery occlusion (15 min.) followed by two hours of recirculation reduced mitochondrial superoxide dismutase (SOD) and glutathione reductase (GR) activities, and increased susceptibility of mitochondrial membranes to in vitro lipid peroxidation in brain regions (i.e., cortex, striatum and hippocampus) of Mongolian gerbil. Intraperitoneal bolus injection (2 mg/kg b.w.) of liposome-entrapped CuZn superoxide dismutase (l-SOD) increased the endogenous SOD activity in normal brain tissue and, when given at the end of ischemia, counteracted both the ischemic reduction of endogenous SOD and the increased peroxidation of mitochondrial membranes. 1-SOD treatment was ineffective in reducing brain swelling, suggesting that superoxide radicals are not a main participant in the process of (post)ischemic brain edema formation.