Condensed Mitotic Chromosome Structure at Nanometer Resolution Using PALM and EGFP- Histones

Photoactivated localization microscopy (PALM) and related fluorescent biological imaging methods are capable of providing very high spatial resolutions (up to 20 nm). Two major demands limit its widespread use on biological samples: requirements for photoactivatable/photoconvertible fluorescent molecules, which are sometimes difficult to incorporate, and high background signals from autofluorescence or fluorophores in adjacent focal planes in three-dimensional imaging which reduces PALM resolution significantly. We present here a high-resolution PALM method utilizing conventional EGFP as the photoconvertible fluorophore, improved algorithms to deal with high levels of biological background noise, and apply this to imaging higher order chromatin structure. We found that the emission wavelength of EGFP is efficiently converted from green to red when exposed to blue light in the presence of reduced riboflavin. The photon yield of red-converted EGFP using riboflavin is comparable to other bright photoconvertible fluorescent proteins that allow <20 nm resolution. We further found that image pre-processing using a combination of denoising and deconvolution of the raw PALM images substantially improved the spatial resolution of the reconstruction from noisy images. Performing PALM on Drosophila mitotic chromosomes labeled with H2AvD-EGFP, a histone H2A variant, revealed filamentous components of ∼70 nm. This is the first observation of fine chromatin filaments specific for one histone variant at a resolution approximating that of conventional electron microscope images (10–30 nm). As demonstrated by modeling and experiments on a challenging specimen, the techniques described here facilitate super-resolution fluorescent imaging with common biological samples.     

Carbonic Anhydrase and Urease Inhibitory Potential of Various Plant Phenolics Using in vitro and in silico Methods

Plant phenolics are known to display many pharmacological activities. In the current study, eight phenolic compounds, e.g., luteolin 5‐O‐β‐glucoside ( 1 ), methyl rosmarinate ( 2 ), apigenin ( 3 ), vicenin 2 ( 4 ), lithospermic acid ( 5 ), soyasaponin II ( 6 ), rubiadin 3‐O‐β‐primeveroside ( 7 ), and 4‐(β‐d ‐glucopyranosyloxy)benzyl 3,4‐dihydroxybenzoate ( 8 ), isolated from various plant species were tested at 0.2 mm against carbonic anhydrase‐II (CA‐II) and urease using microtiter assays. Urease inhibition rate for compounds 1  –  8 ranged between 5.0 – 41.7%, while only compounds 1 , 2 , and 4 showed a considerable inhibition over 50% against CA‐II with the IC₅₀ values of 73.5 ± 1.05, 39.5 ± 1.14, and 104.5 ± 2.50 μm , respectively, where IC₅₀ of the reference (acetazolamide) was 21.0 ± 0.12 μm . In silico experiments were also performed through two docking softwares (Autodock Vina and i‐GEMDOCK) in order to find out interactions between the compounds and CA‐II. Actually, compounds 6 (30.0%) and 7 (42.0%) possessed a better binding capability toward the active site of CA‐II. According to our results obtained in this study, among the phenolic compounds screened, particularly 1 , 2 , and 4 appear to be the promising inhibitors of CA‐II and may be further investigated as possible leads for diuretic, anti‐glaucoma, and antiepileptic agents.     

The first investigation of the diversity of bacteria associated with <Emphasis Type=Italic>Leptinotarsa decemlineata</Emphasis> (Coleoptera: Chrysomelidae)

Colorado potato beetle, Leptinotarsa decemlineata (Say), is a devastating pest of potatoes in North America and Europe. L. decemlineata has developed resistance to insecticides used for its control. In this study, in order to find a more effective potential biological control agent against L. decemlineata, we investigated its microbiota and tested their insecticidal effects. According to morphological, physiological and biochemical tests as well as 16S rDNA sequences, microbiota was identified as Leclercia adecarboxylata (Ld1), Acinetobacter sp. (Ld2), Acinetobacter sp. (Ld3), Pseudomonas putida (Ld4), Acinetobacter sp. (Ld5) and Acinetobacter haemolyticus (Ld6). The insecticidal activities of isolates at 1.8×10⁹ bacteria/mL dose within five days were 100%, 100%, 35%, 100%, 47% and 100%, respectively, against the L. decemlineata larvae. The results indicate that Leclercia adecarboxylata (Ld1) and Pseudomonas putida (Ld4) isolates may be valuable potential biological control agents for biological control of L. decemlineata.     

The first study on the bacterial flora of the european spruce bark beetle, Dendroctonus micans (Coleoptera: Scolytidae)

The European spruce bark beetle, Dendroctonus micans Kugelann (Coleoptera, Scolytidae), is one of the most serious pests of oriental spruce (Picea orientalis L.) in Turkey. In this study, we investigated bacterial flora of D. micans collected from different populations of the forests of Eastern Black Sea Region of Turkey from 2002 to 2004. Seven different bacteria were isolated from healthy, diseased and dead specimens based on the color of colony and morphology. According to morphological, physiological and biochemical properties, metobolic enyzme profile by BIOLOG microtiter plate system, and total cellular fatty acid profile by Microbial Identification System (MIS), isolates were identified as Micrococcus luteus, Bacillus thuringiensis subsp. morrisoni, Serratia grimesii, Enterobacter cloaceae, Enterobacter intermedius, Streptococcus sp. and Pseudomonas putida. This is the first study on the bacterial flora of D. micans.     

Genetic and environmental influences on one-trial conditioned context aversion in mice

Anticipatory nausea (AN) is caused by an association between contextual cues and the experience of nausea (the side effects of chemotherapy or radiation treatment) and it develops predominantly in female patients undergoing chemotherapy. Preclinical studies in rodents show that the administration of an illness-inducing agent in the presence of novel contextual cues can cause conditioned context aversion (CCA) and this has been proposed to model AN. The literature also suggests that brief pre-exposure to a novel context prior to shock delivery is critical in the development of contextual fear conditioning in rodents (a phenomenon known as Immediate Shock Deficit), but this has not been assessed in CCA. The aim of present study was to develop a CCA paradigm to assess this in outbred (CD1) and inbred (C57BL/6J) mice and evaluate potential sex differences. The results revealed that a single conditioning trial in which a distinctive context was paired with LiCl-induced illness was sufficient to elicit a conditioned response in both female and male CD1 outbred mice, but not in C57BL/6J inbred mice. In addition, CCA was facilitated when animals had prior experience with the context. Finally, outbred female mice showed longer and more robust retention of CCA than male mice, which parallels clinical findings. The results indicate the importance of using CD1 outbred mice as an animal model of AN as well as examining sex differences in the CCA paradigm. Similar findings in humans encourage the future use of this novel CCA preclinical mouse model.     

Analysis of the effects of hypothalamic-pituitary-adrenal axis activity in menstrual cycle on ankle proprioception,dynamic balance scores and visual-auditory reaction times in healthy young women

Objectives:Menstrual cycle (MC) can affect not only the female reproductive system, but also functions such as neuromuscular performance. For this reason, the aim of this study is to investigate the effect of hypothalamic-pituitary-adrenal axis (HPA) activity in MC on proprioception, balance and reaction times.Methods:For cortisol analysis, saliva samples were taken from the same women (n=43) in the four phases of MC. While State Trait Anxiety Inventory-I (STAI-I) was applied in each phase to support cortisol analysis, pain was measured with visual analogue scale (VAS). Proprioception, dynamic balance, visual and auditory reaction times (VRT-ART) measurements were made in the four phases of MC.Results:Cortisol, STAI-I and VAS scores, angular deviations in proprioception measurements, dynamic balance scores, VRT and ART measurements were found to show statistically significant difference between MC phases (p<0.05). As a result of the post hoc test conducted to find out which MC phase the statistical difference resulted from, it was found that statistically significant difference was caused by the mensturation (M) phase (p<0.05).Conclusions:It was found that neuromuscular performance and postural control was negatively affected by HPA axis activity in M phase of MC and by pain, which is a significant menstrual symptom.     

Macrophage inflammatory protein-1α: A link between innate immunity and familial mediterranean fever?

The aim of this study is to investigate the relationship between chemokines and the inflammation in Familial Mediterranean Fever (FMF). Forty-nine patients with FMF (41 in remission and 8 in acute attack period) and 20 healthy controls were included in the study. Serum levels of macrophage inflammatory protein-1alpha (MIP-1alpha) were assessed in the patients and the controls, along with other parameters of disease activity, i.e., fibrinogen, C-reactive protein and erythrocyte sedimentation rate. Serum MIP-1alpha levels of the patients with FMF in acute attack period were significantly higher than the patients in remission and healthy controls (p=0.02 and p=0.038, respectively). MIP-1alpha levels were weakly correlated with CRP (r=0.32, p=0.032) levels. MIP-1alpha may have a role in the pathogenesis of FMF attacks. MIP-1alpha and other chemokines may constitute a link between the innate immune system and FMF.     

A three-dimensional approach to mitotic chromosome structure: evidence for a complex hierarchical organization

We describe findings on the architecture of Drosophila melanogaster mitotic chromosomes, made using a three-dimensional-oriented structural approach. Using high-voltage and conventional transmission electron microscopy combined with axial tomography and digital contrast-enhancement techniques, we have for the first time visualized significant structural detail within minimally perturbed mitotic chromosomes. Chromosomes prepared by several different preparative procedures showed a consistent size hierarchy of discrete chromatin structural domains with cross-sectional diameters of 120, 240, 400-500, and 800-1,000 A. In fully condensed, metaphase-arrested chromosomes, there is evidence for even larger-scale structural organization in the range of 1,300-3,000-A size. The observed intrachromosomal arrangements of these higher-order structural domains show that both the radial loop and sequential helical coiling models of chromosome structure are over-simplifications of the true situation. Finally, our results suggest that the pathway of chromatin condensation through mitosis consists of concurrent changes occurring at several levels of chromatin organization, rather than a strictly sequential folding process.     

A library of monoclonal antibodies to nuclear proteins from Drosophila melanogaster embryos. Characterization by a cultured cell assay

To study the structure and function of the cell nucleus, a library of 170 monoclonal antibodies was produced to nuclear antigens from 3-6 h old Drosophila embryos. In preparation for immunization, nuclei were separated, at neutral pH and in the presence of polyamines, into two fractions containing either urea-soluble non-histone nuclear proteins or histones plus small quantities of non-histone proteins complexed to DNA. The antibodies were characterized in a rapid, indirect immunofluorescent assay employing cultured Drosophila cells (Schneider's line 2). Low backgrounds and high specific fluorescence were achieved in this assay by purifying the rhodamine-labelled second antibody on a polystyrene resin and washing the cells with optimal concentrations of detergents. The assay categorized antigens according to their cellular locations: in nuclei, in nuclei plus cytoplasm, or primarily in cytoplasm. A subset of nuclear antigens reacted specifically with the nuclear envelope. In addition, some antibodies were characterized by their reactions with polytene chromosomes. The cultured cell assay provides a new, efficient method for expanding this antibody library. The monoclonal antibodies in the library now provide highly specific tools for investigating structural nuclear proteins and proteins that may be regulatory during embryonic development.