Label-Free Imaging of Lipid Depositions in C. elegans Using Third-Harmonic Generation Microscopy

Elucidation of the molecular mechanisms regulating lipid storage and metabolism is essential for mitigating excess adiposity and obesity, which has been associated with increased prevalence of severe pathological conditions such as cardiovascular disorders and type II diabetes, worldwide. However, imaging fatty acid distribution and dynamics in vivo, at the cellular or organismal level is challenging. We developed a label-free method for visualizing lipid depositions in vivo, based on third harmonic generation (THG) microscopy. THG imaging requires a single pulsed-laser light source, alleviating the technical challenges of implementing coherent anti-Stokes Raman scattering spectroscopy (CARS) to detect fat stores in living cells. We demonstrate that THG can be used to efficiently and reliably visualize lipid droplets in Caenorhabditis elegans. Thus, THG microscopy offers a versatile alternative to fluorescence and dye-based approaches for lipid biology research.     

Interaction of Rhizospheric Bacteria and Nonrhizobial Endophytic Bacteria Moving from the Roots to the Rhizosphere of Pea Plants (Pisum sativum)

Applied Biochemistry and Microbiology - Six of 11 strains of endophytic bacteria from pea (Pisum sativum L.) seeds were found in an aqueous medium of seedling-root growth under hydroculture...     

A leucine----proline mutation in the H1 subdomain of keratin 1 causes epidermolytic hyperkeratosis.

Epidermolytic hyperkeratosis is an autosomal dominant disorder affecting the structural integrity of the suprabasal layers of human epidermis. We have recently documented in one family linkage of the disease phenotype to the cluster of type II keratins. We have now identified a leucine----proline amino acid substitution in the conserved H1 subdomain of keratin 1 that is present only in affected family members. Using a quantitative assay and electron microscopy with synthetic peptides, we show that, whereas the wild-type H1 peptide rapidly disassembles preformed keratin filaments in vitro, the mutant peptide does this far less efficiently. Therefore the mutation in keratin 1 is likely to cause defective keratin filaments and hence a defective cytoskeleton in the epidermal cells in vivo.     

Karyotaxonomische Untersuchungen anPotentilla taurica s.l. in Bulgarie

Several taxa considered by previous authors as intraspecific ofPotentilla taurica s.l. are considered as separate species. A diploid chromosome number 2n=14 and the characteristic chromosomes of karyotypes of 3 Balkan endemicsP. ni?i?i Adam.,P. emilipopii Nyarády andP. pirotensis (Borb.) Mark. are recorded.P. bornmuelleri Borb.,P. mollicrinis (Borb.) Stankov andP. astracanica Jacq. are widespread in South-eastern Europe. Of this groupP. mollicrinis is a diploid (2n=14),P. bornmuelleri is tetraploid and inP. astracanica a polyploid serie of 2x=14, 4x=28, 5x=35 and 6x=42 has been found. The karyotypes of all species are characterized by submetacentric chromosomes.     

Microcalorimetric studies on the lyophilic properties of pectic substances

The exothermic effects observed on wetting pectins with water and aliphatic alcohols were studied using a microcalorimeter.The heat released on wetting 1 g pectin with water was found to be 171 ± 7·5 J g^?1. It was experimentally established that 1 g of dry pectin exothermically bonded up to 0·57 g of water.By using the Gibbs-Helmholtz-Young equation which relates the heat released by wetting to the area of the wetted surface, it was estimated that the surface accessible to water in 1 g of pectin was 1·46 × 10³ m² g^?1. The heat of hydration was independent of the degree of esterification of the pectin. The experimental results revealed that there were about six molecules of energetically bonded water per monomer unit of pectin.A specific interaction between methanol and the methoxyl groups of pectin was observed on wetting pectins with methanol and dependence was established between the released heat and the degree of esterification. No similar dependence was reported for the remaining aliphatic alcohols.     

The development of modified human Hsp70 (HSPA1A) and its production in the milk of transgenic mice

The production of major human heat shock protein Hsp70 (HSPA1A) in a eukaryotic expression system is needed for testing and possible medical applications. In this study, transgenic mice were produced containing wild-type human Hsp70 allele in the vector providing expression in the milk. The results indicated that human Hsp70 was readily expressed in the transgenic animals but did not apparently preserve its intact structure and, hence, it was not possible to purify the protein using conventional isolation techniques. It was suggested that the protein underwent glycosylation in the process of expression, and this quite common modification for proteins expressed in the milk complicated its isolation. To check this possibility, we mutated all presumptive sites of glycosylation and tested the properties of the resulting modified Hsp70 expressed in E. coli. The investigation demonstrated that the modified protein exhibited all beneficial properties of the wild-type Hsp70 and was even superior to the latter for a few parameters. Based on these results, a transgenic mouse strain was obtained which expressed the modified Hsp70 in milk and which was easy to isolate using ATP columns. Therefore, the developed construct can be explored in various bioreactors for reliable manufacture of high quality, uniform, and reproducible human Hsp70 for possible medical applications including neurodegenerative diseases and cancer.     

ERIL1, the plant homologue of ERI‐1, is involved in the processing of chloroplastic rRNAs

Proteins belonging to the enhancer of RNA interference‐1 subfamily of 3′–5′ exoribonucleases participate in divergent RNA pathways. They degrade small interfering RNAs (siRNAs), thus suppressing RNA interference, and are involved in the maturation of ribosomal RNAs and the degradation of histone messenger RNAs (mRNAs). Here, we report evidence for the role of the plant homologue of these proteins, which we termed ENHANCED RNA INTERFERENCE‐1‐LIKE‐1 (ERIL1), in chloroplast function. In vitro assays with AtERIL1 proved that the conserved 3′–5′ exonuclease activity is shared among all homologues studied. Confocal microscopy revealed that ERL1, a nucleus‐encoded protein, is targeted to the chloroplast. To gain insight into its role in plants, we used Nicotiana benthamiana and Arabidopsis thaliana plants that constitutively overexpress or suppress ERIL1. In the mutant lines of both species we observed malfunctions in photosynthetic ability. Molecular analysis showed that ERIL1 participates in the processing of chloroplastic ribosomal RNAs (rRNAs). Lastly, our results suggest that the missexpression of ERIL1 may have an indirect effect on the microRNA (miRNA) pathway. Altogether our data point to an additional piece of the puzzle in the complex RNA metabolism of chloroplasts.