Fluorescent diethylcarbamazine analogues: sites of accumulation in Brugia malayi

New fluorescein and rhodamine B-labeled antifilarial drug DEC analogues for use in drug localization studies with confocal microscopy have been prepared by a high-yield three-step synthesis. The resulting beta-amine-substituted DEC analogue has a single ethyl substituent which is beta-aminated to accommodate the fluorophore of either fluorescein isothiocyananate or rhodamine B. Confocal microscopy is used to show that the drug accumulates in the adult filarial worms in the pharynx, esophagus, and near the nerve ring of all adults, as well as in the uteri and vulva and the testes of the females and males.     

A single proline substitution is critical for the thermostabilization of Clostridium beijerinckii alcohol dehydrogenase

Analysis of the three-dimensional structures of three closely related mesophilic, thermophilic, and hyperthermophilic alcohol dehydrogenases (ADHs) from the respective microorganisms Clostridium beijerinckii (CbADH), Entamoeba histolytica (EhADH1), and Thermoanaerobacter brockii (TbADH) suggested that a unique, strategically located proline residue (Pro100) might be crucial for maintaining the thermal stability of EhADH1. To determine whether proline substitution at this position in TbADH and CbADH would affect thermal stability, we used site-directed mutagenesis to replace the complementary residues in both enzymes with proline. The results showed that replacing Gln100 with proline significantly enhanced the thermal stability of the mesophilic ADH: DeltaT(1/2) (60 min) = + 8 degrees C (temperature of 50% inactivation after incubation for 60 min), DeltaT(1/2) (CD) = +11.5 degrees C (temperature at which 50% of the original CD signal at 218 nm is lost upon heating between 30 degrees and 98 degrees C). A His100 --> Pro substitution in the thermophilic TbADH had no effect on its thermostability. An analysis of the three-dimensional structure of the crystallized thermostable mutant Q100P-CbADH suggested that the proline residue at position 100 stabilized the enzyme by reinforcing hydrophobic interactions and by reducing the flexibility of a loop at this strategic region.     

Life and death in peripheral T cells

During the course of an immune response, antigen-reactive T cells clonally expand and then are removed by apoptosis to maintain immune homeostasis. Life and death of T cells is determined by multiple factors, such as T-cell receptor triggering, co-stimulation or cytokine signalling, and by molecules, such as caspase-8 (FLICE)-like inhibitory protein (FLIP) and haematopoietic progenitor kinase 1 (HPK1), which regulate the nuclear factor-kappaB (NF-kappaB) pathway. Here, we discuss the concepts of activation-induced cell death (AICD) and activated cell-autonomous death (ACAD) in the regulation of life and death in T cells.     

Seed morphology of Rhododendron sichotense (Ericaceae): systematic implications

The size of Rhododendron sichotense seeds and exotesta cells from 20 populations were studied using a scanning electron microscope (SEM). The results of a correlation analysis showed that seed size depends on bioclimatic factors. As a species marker, we used the exotesta cell elongation coefficient. This characteristic was unresponsive to environmental variables, demonstrating its taxonomic significance. In the centre of the species range, the samples had an exotesta cell elongation coefficient that was typical for the species, but in samples from the far northern and southern parts of the range, where the distribution of R. sichotense meets that of of R. dauricum L. and R. mucronulatum Turcz., respectively, the value of the exotesta cell elongation coefficient was intermediate between these species. Therefore, the exotesta cell elongation coefficient can be recommended for use in the detection of hybridisation areas.     

Knockdown of CENH3 in Arabidopsis reduces mitotic divisions and causes sterility by disturbed meiotic chromosome segregation

The histone H3 variant (CENH3) of centromeric nucleosomes is essential for kinetochore assembly and thus for chromosome segregation in eukaryotes. The mechanism(s) that determine centromere identity, assembly and maintenance of kinetochores are still poorly understood. Although the role of CENH3 during mitosis has been studied in several organisms, little is known about its meiotic function. We show that RNAi-mediated CENH3 knockdown in Arabidopsis thaliana caused dwarfism as the result of a reduced number of mitotic divisions. The remaining mitotic divisions appeared to be error-free. CENH3 RNAi transformants had reduced fertility because of frequently disturbed meiotic chromosome segregation. N-terminally truncated EYFP-CENH3(C) is deposited to and functional within Arabidopsis centromeres of mitotic chromosomes, but cannot be loaded onto centromeres of meiotic nuclei. Thus the N-terminal part is apparently required for CENH3 loading during meiosis. EYFP-CENH3(C) expression reduces the amount of endogenous CENH3, thus mimicking the effect of RNAi. The consequences of reduced endogenous CENH3 and lack of meiotic incorporation of EYFP-CENH3(C) are reduced fertility caused by insufficient CENH3 loading to the centromeres of meiotic chromosomes, subsequent lagging of chromosomes and formation of micronuclei.     

Pre-clinical and clinical significance of heparanase in Ewing's sarcoma

Heparanase is an endoglycosidase that specifically cleaves heparan sulphate side chains of heparan sulphate proteoglycans, activity that is strongly implicated in cell migration and invasion associated with tumour metastasis, angiogenesis and inflammation. Heparanase up-regulation was documented in an increasing number of human carcinomas, correlating with reduced post-operative survival rate and enhanced tumour angiogenesis. Expression and significance of heparanase in human sarcomas has not been so far reported. Here, we applied the Ewing's sarcoma cell line TC71 and demonstrated a potent inhibition of cell invasion in vitro and tumour xenograft growth in vivo upon treatment with a specific inhibitor of heparanase enzymatic activity (compound SST0001, non-anticoagulant N-acetylated, glycol split heparin). Next, we examined heparanase expression and cellular localization by immunostaining of a cohort of 69 patients diagnosed with Ewing's sarcoma. Heparanase staining was noted in all patients. Notably, heparanase staining intensity correlated with increased tumour size (P = 0.04) and with patients' age (P = 0.03), two prognostic factors associated with a worse outcome. Our study indicates that heparanase expression is induced in Ewing's sarcoma and associates with poor prognosis. Moreover, it encourages the inclusion of heparanase inhibitors (i.e. SST0001) in newly developed therapeutic modalities directed against Ewing's sarcoma and likely other malignancies.     

Stress-Induced Changes in Optical Properties,Pigment and Fatty Acid Content of <Emphasis Type="Italic">Nannochloropsis</Emphasis> sp.: Implications for Non-destructive Assay of Total Fatty Acids

In order to develop a practical approach for fast and non-destructive assay of total fatty acid (TFA) and pigments in the biomass of the marine microalga Nannochloropsis sp. changes in TFA, chlorophyll, and carotenoid contents were monitored in parallel with the cell suspension absorbance. The experiments were conducted with the cultures grown under normal (complete nutrient f/2 medium at 75 μmol PAR photons/(m² s)) or stressful (nitrogen-lacking media at 350 μmol PAR photons/(m² s)) conditions. The reliable measurement of the cell suspension absorbance using a spectrophotometer without integrating sphere was achieved by deposition of cells on glass–fiber filters in the chlorophyll content range of 3–13 mg/L. Under stressful conditions, a 30–50% decline in biomass and chlorophyll, retention of carotenoids and a build-up of TFA (15–45 % of dry weight) were recorded. Spectral regions sensitive to widely ranging changes in carotenoid-to-chlorophyll ratio and correlated changes of TFA content were revealed. Employing the tight inter-correlation of stress-induced changes in lipid metabolism and rearrangement of the pigment apparatus, the spectral indices were constructed for non-destructive assessment of carotenoid-to-chlorophyll ratio (range 0.3–0.6; root mean square error (RMSE) = 0.03; r ² = 0.93) as well as TFA content of Nannochloropsis sp. biomass (range 5.0–45%; RMSE = 3.23 %; r ² = 0.89) in the broad band 400–550 nm normalized to that in chlorophyll absorption band (centered at 678 nm). The findings are discussed in the context of real-time monitoring of the TFA accumulation by Nannochloropsis cultures under stressful conditions.     

The tomato early fruit specific gene <Emphasis Type="Italic">Lefsm1</Emphasis> defines a novel class of plant-specific SANT/MYB domain proteins

We describe here a novel plant-specific gene, Lefsm1 (fruit SANT/MYB-like 1) harboring a single SANT/MYB domain. The expression of Lefsm1 is specific to the very early stages of tomato (Lycopersicon esculentum) fruit development. Ectopic expression of Lefsm1 results in severe developmental alterations manifested in retarded growth, and reduced apical dominance during tomato and Arabidopsis seedling development. A promoter sequence residing 1.0 kb upstream to the translation initiation codon confers the organ-specific expression of the gene. Lefsm1 belongs to a novel small gene family consisting of five to six members in tomato, Arabidopsis and rice. The SANT/MYB domain of LeFSM1 and its orthologs in Arabidopsis and rice differs from that of all other plant or animal MYB proteins and from the SANT domains found in part of the chromatin remodeling proteins. Together, our results indicate that Lefsm1 is a founding member of a small family of proteins containing a novel MYB/SANT domain which is likely to participate in the regulation of a plant-specific developmental program.