Human erythrocyte flickering: temperature,ATP concentration,water transport,and cell aging,plus a computer simulation

Images of human erythrocytes from a healthy donor were recorded under differential interference contrast (DIC) microscopy; they were acquired rapidly (~336 Hz) and the intensity of the centermost pixel of each cell was recorded for ~60 s (20,000 values). Various techniques were used to analyze the data, including detrended fluctuation analysis (DFA) and multiscale entropy (MSE); however, power spectrum analysis was deemed the most appropriate for metrifying and comparing results. This analysis was used to compare cells from young and old populations, and after perturbing normal conditions, with changes in temperature, adenosine triphosphate (ATP) concentration (using NaF, an inhibitor of glycolysis, and α-toxin, a pore-forming molecule used to permeabilize red cells to ATP), and water transport rates [using glycerol, and p-chloromercuriphenylsulfonic acid (pCMBS) to inhibit aquaporins, AQPs]. There were measurable differences in the membrane fluctuation characteristics in populations of young and old cells, but there was no significant change in the flickering time series on changing the temperature of an individual cell, by depleting it of ATP, or by competing with the minor water exchange pathway via AQP3 using glycerol. However, pCMBS, which inhibits AQP1, the major water exchange pathway, inhibited flickering in all cells, and yet it was restored by the membrane intercalating species dibutyl phthalate (DBP). We developed a computer model to simulate acquired displacement spectral time courses and to evaluate various methods of data analysis, and showed how the flexibility of the membrane, as defined in the model, affects the flickering time course.     

Identification of the protease responsible for the antiadhesive properties of dog blood serum

The antiadhesive effect of dog blood serum described previously was shown to be associated with the proteolytic activity of the serum components. A protease exhibiting this antiadhesive effect was isolated by several fractionation stages and identified with plasmin.     

Josef Rudinger Memorial Lecture: Use of peptides to modulate protein‐protein interactions

Peptides are destined to play a major role as therapeutic agents. My laboratory is contributing to speeding up this process. On the one hand, we devote efforts to studying the molecular details and dynamics of the events that occur during molecular recognition at protein surfaces. We succeeded to design and synthesize peptides able to modulate these recognition events either permanently or in response to light. On the other hand, we are discovering and designing peptides able to cross biological barriers. Our aim is to use these peptides as shuttles for targeting therapeutic agents to organs, tissues, or cells, with a special emphasis on drug delivery to the brain. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Identification of P‐glycoprotein co‐fractionating proteins and specific binding partners in rat brain microvessels

Drug delivery to the brain for the treatment of pathologies with a CNS component is a significant clinical challenge. P‐glycoprotein (PgP), a drug efflux pump in the endothelial cell membrane, is a major factor in preventing therapeutics from crossing the blood‐brain barrier (BBB). Identifying PgP regulatory mechanisms is key to developing agents to modulate PgP activity. Previously, we found that PgP trafficking was altered concomitant with increased PgP activity and disassembly of high molecular weight PgP‐containing complexes during acute peripheral inflammatory pain. These data suggest that PgP activity is post‐translationally regulated at the BBB. The goal of the current study was to identify proteins that co‐localize with PgP in rat brain microvessel endothelial cell membrane microdomains and use the data to suggest potential regulatory mechanisms. Using new density gradients of microvessel homogenates, we identified two unique pools (1,2) of PgP in membrane fractions. Caveolar constituents, caveolin1, cavin1, and cavin2, co‐localized with PgP in these fractions indicating the two pools contained caveolae. A chaperone (Hsc71), protein disulfide isomerase and endosomal/lysosomal sorting proteins (Rab5, Rab11a) also co‐fractionated with PgP in the gradients. These data suggest signaling pathways with a potential role in post‐translational regulation of PgP activity at the BBB.

     

Limits of application of initiated chemiluminescence in monitoring of oncological process of mucous membrane of mouth and larynx

Investigation into the limits of application of chemiluminescence (CL) methods in oncology still attracts the attention of researchers. In the present work we analyze the screening and monitoring of oncological processes (OP) in the mucous membrane of the mouth and larynx by initiated CL (ICL). Chemiluminescence has already been used by stomatologists to define the start of OP, but methods that reflect the metabolic changes in organism under cancer diagnostics still have not found their place. This work presents results of ICL on blood serum (BS) of patients with oncological diseases at different stages of medical treatment compared with those of healthy people. We found an essential metabolic difference only in types of OP that are characterized by two maxima on chemiluminograms. These OP represent only 12.81% of groups of patients with oncological diseases. The possibility to apply ICL methods to monitor operation quality and control medical treatment at different stages when the two ICL maxima are present is established. At present, the chemiluminograms with the two maxima are mostly informative, but this does not exclude the quantitative analysis of other ICL kinetic methods and is encouraging for their investigation. Any OP introduces changes in organism function and these should be reflected in the ICL. Copyright © 2016 John Wiley & Sons, Ltd.     

Enhancement of Astragalus polysaccharide on the immune responses in pigs inoculated with foot-and-mouth disease virus vaccine

The effects of Astragalus polysaccharides (APS) on the immune response in pigs immunized with foot-and-mouth disease virus (FMDV) vaccine were investigated. Fifteen pigs were randomly divided into five groups. Four groups were vaccinated with a FMDV inactivated vaccine. Pigs in three experimental groups were administered varying doses of APS (APS1, 5 mg/kg; APS2, 10 mg/kg; APS3, 20 mg/kg). The influence of APS on the number of CD3⁺CD4⁻CD8⁺ cytotoxic T cells, CD3⁺CD4⁺CD8⁺ T helper memory cells, and CD3⁻CD4⁻CD8⁺ natural killer cells among peripheral blood lymphocytes (PBL) in the three APS groups were significant compared to the vaccine group. In vitro stimulation of PBL by Con A and LPS in APS groups induced a stronger proliferative response at 2 and 6 weeks post-inoculation (PI). APS markedly increased the titer of FMDV-specific antibody in a dose-dependent manner, and up-regulated mRNA expression of IFN-γ and IL-6. APS could potentially be used as an immunomodulator for a FMDV vaccine and provide better protection against FMDV.     

Does the niche breadth or trade‐off hypothesis explain the abundance–occupancy relationship in avian Haemosporidia?

Two hypotheses have been proposed to explain the abundance–occupancy relationship (AOR) in parasites. The niche breadth hypothesis suggests that host generalists are more abundant and efficient at colonizing different host communities than specialists. The trade‐off hypothesis argues that host specialists achieve high density across their hosts' ranges, whereas generalists incur the high cost of adaptation to diverse immuno‐defence systems. We tested these hypotheses using 386 haemosporidian cytochrome‐b lineages (1894 sequences) recovered from 2318 birds of 103 species sampled in NW Africa, NW Iberia, W Greater Caucasus and Transcaucasia. The number of regions occupied by lineages was associated with their frequency suggesting the presence of AOR in avian Haemosporidia. However, neither hypothesis provided a better explanation for the AOR. Although the host generalist Plasmodium SGS1 was over three times more abundant than other widespread lineages, both host specialists and generalists were successful in colonizing all study regions and achieved high overall prevalence.     

Effect of zinc supplementation from different sources on growth, nutrient digestibility, blood metabolic profile, and immune response of male guinea pigs

Forty weaned male guinea pigs of 208.20±6.62 g mean body weight were divided into 4 groups of 10 animals in a randomized block design. All of the guinea pigs were fed a basal diet [25% ground maize hay, 30% ground maize grain, 22% ground chickpea (Cicer arietinum L.), 9.5% deoiled rice bran, 6% soybean meal, 6% fish meal, 1.45% mineral supplement (without Zn) and 0.05% ascorbic acid] and available green fodder. Group I served as the control (no Zn supplementation), whereas 20 ppm Zn was added in the diet in groups II, III, and IV either as zinc sulfate (ZnSO₄), zinc amino acid complex (ZAAC), and ZnSO₄ ⁺ ZAAC in equal parts, respectively. Experimental feeding lasted for 70 d, including a 3-d digestibility trial. Blood was collected through cardiac puncture from four animals in each group at d 0 and subsequently at the end of experimental feeding. After 40 d of experimental feeding, four animals from each group were injected with 0.4 mL of Brucella abortus cotton strain-19 vaccine to assess the humoral immune response of the animals. After 10 wk of study, four animals from each group were sacrificed to study the concentration of Zn, Cu, Co, Fe, and Mn in the liver, pancreas and spleen. Results revealed no significant difference in the feed intake, body weight gain, and digestibility of the nutrients, except for crude protein (CP) digestibility, which was significantly (p<0.05) lower in group IV. Although concentrations of serum glucose, Ca, and P and the albumin:globulin (A:G) ratio were similar in the different groups, the total protein, albumin, and serum alkaline phosphatase activity were higher in all of the Zn-supplemented groups on d 70. The serum Zn levels at the end of experimental feeding were significantly higher in groups II and III, whereas serum Mn levels were found to be significantly (p<0.05) higher in groups III and IV. The organ weights (as percentage of body weights) did not show any differences among the treatment groups. Although the Mn concentration was significantly (p<0.05) higher in the pancreas, the Cu concentration was significantly (p<0.05) reduced in the spleen in all of the Zn-supplemented groups. The humoral immune response (antibody titer values) on d 14 of vaccination was significantly (p<0.05) higher in all of the Zn-supplemented groups. It was concluded that the 20-ppm level of Zn in the diet might be adequate for growth and nutrient utilization in guinea pigs, but supplementation of 20-ppm zinc significantly improved the immune response and impact was more prominent with the ZAAC (organic source) compared to ZnSO₄ (inorganic source).     

Novel selenoorganic compounds as modulators of oxidative stress in blood platelets

Many selenoorganic compounds play an important role in biochemical processes and act as antioxidants, enzyme inhibitors, or drugs. The effects of five new synthesized selenoorganic compounds (2-(5-chloro-2-pyridyl)-7-azabenzisoselenazol-3(2H)-one; 2-phenyl-7-azabenzisoselenazol-3(2H)-one; 2-(pyridyl)-7-azabenzisoselenazol-3(2H)-one; 7-azabenzisoselenazol-3(2H)-one; bis(2-aminophenyl) diselenide) on oxidative changes in human blood platelets and in plasma were studied in vitro and compared with those of ebselen, a well known antioxidant. Our studies demonstrated that bis(2-aminophenyl) diselenide has distinctly protective effects against oxidative stress in blood platelets and in plasma. It might have greater biological relevance and stronger pharmacological effects than ebselen.