A Nanoscale Modification of Materials at Thermoplasmonic Laser-Induced Backside Wet Etching of Sapphire

Plasmonics - Laser-induced backside wet etching using water solution of plasmon precursor (AgNO3) as an absorbing medium provides effective structuring of such a complex material as sapphire. At...     

Skeletal Muscle Insulin Resistance Associated with Cholesterol-Induced Activation of Macrophages Is Prevented by High Density Lipoprotein

Background

Emerging evidence suggests that high density lipoprotein (HDL) may modulate glucose metabolism through multiple mechanisms including pancreatic insulin secretion as well as insulin-independent glucose uptake into muscle. We hypothesized that HDL may also increase skeletal muscle insulin sensitivity via cholesterol removal and anti-inflammatory actions in macrophages associated with excess adiposity and ectopic lipid deposition.

Methods

Human primary and THP-1 macrophages were treated with vehicle (PBS) or acetylated low density lipoprotein (acLDL) with or without HDL for 18 hours. Treatments were then removed, and macrophages were incubated with fresh media for 4 hours. This conditioned media was then applied to primary human skeletal myotubes derived from vastus lateralis biopsies taken from patients with type 2 diabetes to examine insulin-stimulated glucose uptake.

Results

Conditioned media from acLDL-treated primary and THP-1 macrophages reduced insulin-stimulated glucose uptake in primary human skeletal myotubes compared with vehicle (primary macrophages, 168±21% of basal uptake to 104±19%; THP-1 macrophages, 142±8% of basal uptake to 108±6%; P<0.05). This was restored by co-treatment of macrophages with HDL. While acLDL increased total intracellular cholesterol content, phosphorylation of c-jun N-terminal kinase and secretion of pro- and anti-inflammatory cytokines from macrophages, none were altered by co-incubation with HDL. Insulin-stimulated Akt phosphorylation in human skeletal myotubes exposed to conditioned media was unaltered by either treatment condition.

Conclusion

Inhibition of insulin-stimulated glucose uptake in primary human skeletal myotubes by conditioned media from macrophages pre-incubated with acLDL was restored by co-treatment with HDL. However, these actions were not linked to modulation of common pro- or anti-inflammatory mediators or insulin signaling via Akt.     

Enzyme immunoassay of (24R)-brassinosteroids

Brassinosteroids are a new group of phytohormones that are widely distributed in plants and play an important role in the processes of plant growth and development. Physiological concentrations of brassinosteroids in plants are extremely low, and their analysis in organs and tissues is very difficult. This study is devoted to the chemical aspects of elaboration and to bioanalytical parameters of an immunoenzymatic system for quantitative determination of the phytohormones 24-epicastasterone and 24-epibrassinolide.     

The humoral immunity against diphtheria

Results of the conducted study showed that naturally acquired antibacterial and postvaccinal antitoxic antibodies against diphtheria were found in human blood sera. Challenge of ADT-M toxoid to adults resulted in production of antitoxic as well as antibacterial antibodies in high concentrations. In response to challenge of ADT-M toxoid simultaneously with bacterial vaccine against diphtheria Codivac both antibacterial and antitoxic antibodies were synthesized in blood on optimal physiologic levels. This study revealed dynamics of some specific characteristics of humoral immune response after challenge of two different vaccines against diphtheria--ADT-M toxoid and Codivac vaccine.     

Transformation of the dermal collagen framework under laser heating

The aim of this study was to compare between the changes undergone by the dermal collagen framework when heated by IR laser radiation and by traditional means and to reveal the specific features of the dermal matrix modification under moderate IR laser irradiation. Rabbit skin specimens were heated to 50°C, 55°C, 60°C and 65°C in a calorimeter furnace and with a 1.68‐μm fiber Raman laser. The proportion of the degraded collagen macromolecules was determined by differential scanning calorimetry. Changes in the architectonics of the collagen framework were revealed by using standard, phase‐contrast, polarization optical and scanning electron microscopy techniques. The collagen denaturation and dermal matrix amorphization temperature in the case of laser heating proved to be lower by 10°C than that for heating in the calorimeter furnace. The IR laser treatment of the skin was found to cause a specific low‐temperature (45°C‐50°C) transformation of its collagen framework, with some collagen macromolecules remaining intact. The transformation reduces to the splitting of collagen bundles and distortion of the course of collagen fibers. The denaturation of collagen macromolecules in the case of traditional heating takes its course in a threshold manner, so that their pre‐denaturation morphological changes are insignificant.     

Prion Infection Impairs Cholesterol Metabolism in Neuronal Cells

Conversion of prion protein (PrP^C) into a pathological isoform (PrP^Sc) during prion infection occurs in lipid rafts and is dependent on cholesterol. Here, we show that prion infection increases the abundance of cholesterol transporter, ATP-binding cassette transporter type A1 (ATP-binding cassette transporter type A1), but reduces cholesterol efflux from neuronal cells leading to the accumulation of cellular cholesterol. Increased abundance of ABCA1 in prion disease was confirmed in prion-infected mice. Mechanistically, conversion of PrP^C to the pathological isoform led to PrP^Sc accumulation in rafts, displacement of ABCA1 from rafts and the cell surface, and enhanced internalization of ABCA1. These effects were abolished with reversal of prion infection or by loading cells with cholesterol. Stimulation of ABCA1 expression with liver X receptor agonist or overexpression of heterologous ABCA1 reduced the conversion of prion protein into the pathological form upon infection. These findings demonstrate a reciprocal connection between prion infection and cellular cholesterol metabolism, which plays an important role in the pathogenesis of prion infection in neuronal cells.     

Enhancing apolipoprotein A-I-dependent cholesterol efflux elevates cholesterol export from macrophages in vivo

Eight proteins potentially involved in cholesterol efflux [ABCA1, ABCG1, CYP27A1, phospholipid transfer protein (PLTP), scavenger receptor type BI (SR-BI), caveolin-1, cholesteryl ester transfer protein, and apolipoprotein A-I (apoA-I)] were overexpressed alone or in combination in RAW 264.7 macrophages. When apoA-I was used as an acceptor, overexpression of the combination of ABCA1, CYP27A1, PLTP, and SR-BI (Combination I) enhanced the efflux by 4.3-fold. It was established that the stimulation of efflux was due to increased abundance of ABCA1 and increased apoA-I binding to non-ABCA1 sites on macrophages. This combination caused only a small increase of the efflux to isolated HDL. When HDL was used as an acceptor, overexpression of caveolin-1 or a combination of caveolin-1 and SR-BI (Combination II) was the most active, doubling the efflux to HDL, without affecting the efflux to apoA-I. When tested in the in vivo mouse model of cholesterol efflux, overexpression of ABCA1 and Combination I elevated cholesterol export from macrophages to plasma, liver, and feces, whereas overexpression of caveolin-1 or Combination II did not have an effect. We conclude that pathways of cholesterol efflux using apoA-I as an acceptor make a predominant contribution to cholesterol export from macrophages in vivo.     

Effectivity of expression of mature forms of mutant human apolipoprotein A-I.

In order to probe the structural and functional properties of a central region of apolipoprotein A-I (apoA-I), we engineered mutants of the mature form of the protein and expressed them using the baculovirus/insect cell expression system. The mutations which targeted the region of apoA-I between amino acids 140 and 150 included: (i) deletion of the region 140-150 (apoA-I(Delta140-150)); (ii) substitution of arginine 149 with valine (apoA-I(R149V)); (iii) substitution of proline 143 with alanine (apoA-I(P143A)); (iv) deletion of region 63-73 (apoA-I(Delta63-73)), which has structural properties similar to 140-150; and (v) a chimeric protein substituting amino acids 140-150 with amino acids 63-73 (apoA-I(140-150 --> 63-73)). The efficiencies of synthesis were vastly different for the various mutants as follows: apoA-I(R149V) > apoA-I(140-150 --> 63-73) > apoA-I(Delta63-73) > apoA-I(P143A) > apoA-I > apoA-I(Delta140-150). About 50% of the synthesized wild type and all apoA-I mutants was retained in the cells. During expression of apoA-I(R149V) an unusual spontaneous recombination occurred. In addition to the expected mutant, another form of apoA-I with an apparent M(r) of 36K was produced which consisted of a duplication of the amino-terminal end of apoA-I, from the prepeptide through to amino acid 62, linked to the original pre-apoA-I(R149V) sequence via a 4-amino-acid linker. Despite the fact that this form of apoA-I carries two prepeptides and consequently two cleavage sites, there was little, if any, cleavage at the internal cleavage site. During expression, less than 20% of this mutant was retained in the cells. These results demonstrate that at least in the model of insect cells, the efficiency of apoA-I synthesis, processing, and secretion depends on apoA-I secondary structure and/or folding.     

Demethylation using the epigenetic modifier, 5-azacytidine, increases the efficiency of transient transfection of macrophages

This study was aimed at developing a method for high-efficiency transient transfection of macrophages. Seven methods were evaluated for transient transfection of murine macrophage RAW 264.7 cells. The highest transfection efficiency was achieved with DEAE-dextran, although the proportion of cells expressing the reporter gene did not exceed 20%. It was subsequently found that the cytomegalovirus plasmid promoter in these cells becomes methylated. When cells were treated with the methylation inhibitor 5-azacytidine, methylation of the plasmid promoter was abolished and a dose-dependent stimulation of reporter gene expression was observed with expression achieved in more than 80% of cells. Treatment of cells with 5-azacytidine also caused increased efficiency of transfection of macrophages with plasmids driven by RSV, SV40, and EF-1alpha promoters and transient transfection of human HepG2 cells. Inhibition of methylation also increased the amount and activity of sterol 27-hydroxylase (CYP27A1) detected in RAW 264.7 cells transfected with a CYP27A1 expression plasmid. Treatment of cells with 5-azacytidine alone did not affect either cholesterol efflux from nontransfected cells or expression of ABCA1 and CYP27A1. However, transfection with CYP27A1 led to a 2- to 4-fold increase of cholesterol efflux. We conclude that treatment with 5-azacytidine can be used for high-efficiency transient transfection of macrophages.