A new multimodal confocal microscope has been developed, which includes a parallel Partial Wave Spectroscopic (PWS) microscopy path. This combination of modalities allows molecular‐specific sensing of nanoscale intracellular structure using fluorescent labels. Combining molecular specificity and sensitivity to nanoscale structure allows localization of nanostructural intracellular changes, which is critical for understanding the mechanisms of diseases such as cancer. To demonstrate the capabilities of this multimodal instrument, we imaged HeLa cells treated with valinomycin, a potassium ionophore that uncouples oxidative phosphorylation. Colocalization of fluorescence images of the nuclei (Hoechst 33342) and mitochondria (anti‐mitochondria conjugated to Alexa Fluor 488) with PWS measurements allowed us to detect a significant decrease in nuclear nanoscale heterogeneity (Σ), while no significant change in Σ was observed at mitochondrial sites. In addition, application of the new multimodal imaging approach was demonstrated on human buccal samples prepared using a cancer screening protocol. These images demonstrate that nanoscale intracellular structure can be studied in healthy and diseased cells at molecular‐specific sites.
Although insect herbivores are known to evolve resistance to insecticides through multiple genetic mechanisms, resistance in individual species has been assumed to follow the same mechanism. While both mutations in the target site insensitivity and increased amplification are known to contribute to insecticide resistance, little is known about the degree to which geographic populations of the same species differ at the target site in a response to insecticides. We tested structural (e.g., mutation profiles) and regulatory (e.g., the gene expression of Ldace1 and Ldace2, AChE activity) differences between two populations (Vermont, USA and Belchow, Poland) of the Colorado potato beetle, Leptinotarsa decemlineata in their resistance to two commonly used groups of insecticides, organophosphates, and carbamates. We established that Vermont beetles were more resistant to azinphos‐methyl and carbaryl insecticides than Belchow beetles, despite a similar frequency of resistance‐associated alleles (i.e., S291G) in the Ldace2 gene. However, the Vermont population had two additional amino acid replacements (G192S and F402Y) in the Ldace1 gene, which were absent in the Belchow population. Moreover, the Vermont population showed higher expression of Ldace1 and was less sensitive to AChE inhibition by azinphos‐methyl oxon than the Belchow population. Therefore, the two populations have evolved different genetic mechanisms to adapt to organophosphate and carbamate insecticides. 相似文献
T4 RNA ligase has been shown to synthesize nucleoside and dinucleoside 5'-polyphosphates by displacement of the AMP from the E-AMP complex with polyphosphates and nucleoside diphosphates and triphosphates. Displacement of the AMP by tripolyphosphate (P3) was concentration dependent, as measured by SDS/PAGE. When the enzyme was incubated in the presence of 0.02 mm [alpha-32P] ATP, synthesis of labeled Ap4A was observed: ATP was acting as both donor (Km, microm) and acceptor (Km, mm) of AMP from the enzyme. Whereas, as previously known, ATP or dATP (but not other nucleotides) were able to form the E-AMP complex, the specificity of a compound to be acceptor of AMP from the E-AMP complex was very broad, and with Km values between 1 and 2 mm. In the presence of a low concentration (0.02 mm) of [alpha-32P] ATP (enough to form the E-AMP complex, but only marginally enough to form Ap4A) and 4 mm of the indicated nucleotides or P3, the relative rate of synthesis of the following radioactive (di)nucleotides was observed: Ap4X (from XTP, 100); Ap4dG (from dGTP, 74); Ap4G (from GTP, 49); Ap4dC (from dCTP, 23); Ap4C (from CTP, 9); Ap3A (from ADP, 5); Ap4ddA, (from ddATP, 1); p4A (from P3, 200). The enzyme also synthesized efficiently Ap3A in the presence of 1 mm ATP and 2 mm ADP. The following T4 RNA ligase donors were inhibitors of the synthesis of Ap4G: pCp > pAp > pA2'p. 相似文献
Habitat preferences by Houbara Bustards Chlamydotis [undulata] macqueenii in Harrat al-Harrah reserve, in northern Saudi Arabia were determined from sightings of birds in all seasons over three years. Vegetation and crawling invertebrate abundance were sampled in each habitat. Houbara Bustards showed seasonally changing habitat preferences that appeared to be influenced primarily by vegetation phenology, abundance and cover. More densely vegetated areas (10–17% cover) were preferred. Seasonal and inter-habitat variations in invertebrate numbers were not reflected in differential habitat use by Houbara Bustards. The highest selection ratio for a single habitat (dry lakes) occurred in summer, coinciding with the fruiting of Shafallah Capparis spinosa. Selectivity of habitats was least in spring, when green vegetation was most widespread. Changes in Houbara Bustard habitat preferences in response to marked seasonal changes in habitats brought about by well-defined patterns of rainfall indicate that studies of habitat selection should consider the entire annual cycle. The importance of vegetative cover and the sensitivity of Houbara Bustards to human disturbance suggest that reserves set aside for Houbara Bustards should be extensive, diverse and largely free of livestock, human occupation and its associated disturbances. 相似文献
In Saccharomyces cerevisiae, nutrient limitation stimulates diploid cells to undergo DNA replication and meiosis, followed by the formation of four haploid
spores. Septins are a family of proteins that assemble a ring structure at the mother-daughter neck during vegetative growth,
where they control cytokinesis. In sporulating cells, the septin ring disassembles and septins relocalize to the prospore
membrane. 相似文献
The unusual heterodimeric leishmanial DNA topoisomerase IB consists of a large subunit containing the phylogenetically conserved
"core" domain, and a small subunit harboring the C-terminal region with the characteristic tyrosine residue in the active
site. RNAi silencing of any of both protomers induces a non-viable phenotype in the hemoflagelateTrypanosoma brucei. Unfortunately, this approach is not suitable inLeishmaniawhere gene replacement with an antibiotic marker is the only approach to generate lack-of-function mutants. In this work,
we have successfully generated null mutants in the small subunit of theL. majorDNA topoisomerase IB using two selection markers, each conferring resistance to hygromycin B and puromycin, respectively. 相似文献