A circular dichroism study of human erythrocyte ghost proteins during exposure to 2450 MHz microwave radiation

The effect of 2450 MHz microwave radiation on the proteins of human erythrocyte ghosts has been investigated using circular dichroism spectroscopy. A specially constructed waveguide inserted into the spectropolarimeter allowed the continuous recording of optical activity before, during and after microwave irradiation. The data indicate that high levels of microwave radiation (600 mW/g, specific absorption rate) induce decreases in alpha-helical conformation that may result from both thermal vibrations and increased strain on the intramolecular hydrogen bonds that maintain secondary structure. The latter effect may result from differential intramolecular interactions with the oscillating electric field. Spectrin (bands 1 and 2) isolated from the ghosts was more sensitive to microwave irradiation than intact ghosts, and spectrin-depleted vesicles were the least sensitive. The data, therefore, indicate that the alpha-helical conformation of spectrin is altered by high levels of microwave radiation.     

A novel method for the study of fluorescent probes in biological material during exposure to microwave radiation

Instrumentation has been developed which allows the monitoring of fluorescnece in erythrocyte ghost membranes before, during, and after exposure to microwave radiation. Using non-fluorescent, UV-transmitting transmitting fiber optic cables, excitation light of specific wavelengths was delivered to a stirred sample undergoing irradiation (2450 MHz, CW) within a fluid-filled, temperature-controlled waveguide. Fluorescence was collected using an identical cable and transferred through appropriate filters to standard detecting, amplification and recording devices. We have used the fluorescent probe, 1-anilino-8-naphthalene sulfonate (ANS)_to monitor the effect of mirowave radiation on the binding of calcium to erthrocyte ghosts. Microwave radiation at specific absorption rates of 10 and 200 mW/g had no effect on the binding of ANS to the membranes. Dose-responses curves also showed no influence of microwaves on calcium binding between 2.0 and 10.0 · 10^?4 M. In addition, experiments studying fluorescence energy transfer between intrinsic tryptophan residues and membrane bound ANS showed that intermolecular distance between donor and acceptor are also unaffected by microwave radiation. We have thus shown that 2450 MHz microwve radition at the specific absorption rates studies rates used does not interfere with the binding of calcium to erythrocyte ghosts or alter intermolecular distances intrinsic molecules and bound ANS.     

Influence of CW microwave radiation on in vitro release of enzymes from retinol-treated hepatic lysosomes

Hepatic lysosomes were exposed in vitro to microwave radiation (2450 MHz) either prior to or simultaneously with treatment with retinol (vitamin A), and the release of the lysosomal enzymes, β-glucuronidase, acid phosphatase, and cathepsin D, determined. A 60-min microwave exposure (10 or 100 mW/g) of retinol-treated lysosomes had no effect on the amount of release of β-glucuroni-dase, cathepsin D, or acid phosphatase. In addition, 10 and 100 mW/g irradiation of lysosome fractions for 40 min prior to a 20-min retinol and microwave treatment, had no influence on the release of these enzymes. Finally, the effect of microwave radiation on the loss of latency of acid phosphatase and β-glucuronidase from retinol-treated lysosomes was determined. Microwave radiation had no influence on the rate of appearance of these enzymes in the suspending medium. The results indicate that microwave radiation had no effect on the retinol-induced lysosomal enzyme release.     

The effect of 2450 MHz microwave radiation on histamine secretion by rat peritoneal mast cells

Isolated rat peritoneal mast cells actively secrete histamine in response to reaginic or chemical stimulation. Mast cells were irradiated in a waveguide microwave exposure chamber at 2450 MHz with power absorptions of 8.2 and 41.0 mW/g for periods up to 3 h. These levels of microwave absorption caused no change in the morphological characteristics or viability of the cells. Irradiated mast cells were stimulated with compound 48/80, a potent, noncytotoxic histamine releasing agent. The dose response curves showed that neither prior nor simultaneous irradiation of mast cells at 37°C affected 48/80-induced secretion. However, microwave power absorptions of 41.0 mW/g inhibited secretion at 44.0°C. Precise measurements of the effect of heat on secretion indicated that this level of inhibition could have been produced by a radiation induced increase in cell temperature between 0.4 and 0.9°C above ambient levels. Alternatively, the heat stress produced at 44°C may have sensitized the cells to the electromagnetic effects of the microwave radiation. Rat peritoneal mast cells can therefore be useful as a model for the study of functioning secretory cells during microwave irradiation and can also be used to monitor the synergistic effects of cell heating during in vitro exposure.     

Complement pores in erythrocyte membranes: Analysis of C8/C9 binding required for functional membrane damage

The number of membrane-bound terminal complement proteins (C5b-9) required to generate a functional pore in the human erythrocyte membrane ghost has been determined. Resealed erythrocyte ghost membranes (ghosts) were treated with human complement proteins C5b6, C7, ¹³¹I-C8, and ¹²⁵I-C9 under non-lytic conditions. Following C5b-9 assembly, sucrose-permeant ghosts were separated from C5b-9 ghosts that remained impermeant to sucrose by centrifugation over density barriers formed of 43% (w/v) sucrose. Analysis of ¹³¹I-C8 and ¹²⁵I-C9 bound to sucrose-permeant and sucrose-impermeant subpopulations of C5b-9 ghosts revealed: 1. Sucrose-permeant C5b-9 ghosts show increased uptake of both ¹³¹I-C8 and ¹²⁵I-C9 as compared to ghosts that remain impermeant to sucrose. Ghosts with less than 300 molecules ¹³¹I-C8 bound remain impermeant to sucrose, irrespective of the total C9 input, or, the multiplicity of C9 uptake by membrane C5b-8. 2. In the presence of excess ¹²⁵I-C9, the ratio of ¹²⁵I-C9/¹³¹I-C8 bound to membrane C5b67 is 3.2 ± 0.8 (mean ± 2 S.D.), suggesting an average stoichiometry of 3 C9 per C5b-8. Under these conditions, the ratio of ¹²⁵I-C9/¹³¹I-C8 bound to sucrose-permeant ghosts (3.3 ± 0.7) does not significantly differ from the ratio bound to sucrose-impermeant ghosts (2.9 ± 0.6). 3. With limiting C9 input, the threshold of total C5b-8 uptake required for sucrose permeability increases significantly above 300 per cell when the ratio of bound ¹²⁵I-C9/¹³¹I-C8 is decreased below unity. In the complete absence of C9, 11 700 C5b-8 complexes are bound to sucrose-permeant ghosts. It is concluded that more than 300 C5b-9 complexes must bind to the human erythrocyte to form a sucrose-permeant lesion. Although the binding of one C9 per C5b-8 is critical to the pore-forming activity of these proteins, the binding of additional molecules of C9 to each complex (C9/C8 > 1) does not significantly alter the threshold of total C5b-9 uptake required for lesion formation.     

Effects of microwave radiation (340 and 900 MHz) on different structural levels of erythrocyte membranes

By use of fluorescence probes 1-anilinonaphthalene-8-sulfonic acid, 2-toluidinylnaphthalene-6-sulfonate, pyrene, perylene and chemical label phosphatidylethanolamine 2,4,6-trinitrobenzele sulfonic acid, the effect of microwave radiation on the erythrocyte membrane was studied. The studies with the fluorescence probes were carried out on erythrocyte ghosts and with 2,4,6-trinitrobenzene sulfonic acid on whole erythrocytes. The fluorescence was measured during irradiation of the membranes with 340-MHz microwaves at an SAR of 100 W/kg. Trinitrophenylation of phosphatidylethanolamine from whole erythrocytes was performed simultaneously with microwave irradiation at 900 MHz (10 mW/cm2). It was shown that the microwave field decreased lipid viscosity, altered the structural state of lipid-protein contact regions, and decreased the protein shielding of lipids. These changes corresponded to those produced by thermal action of microwaves.     

Chemistry of the “molecular trap” of protease-catalyzed splicing reaction of complementary segments of α-subunit of hemoglobin A

The complementary fragments of human Hb α, α_1–30, and α_31–141 are spliced together by V8 protease in the presence of 30%n-propanol to generate the full-length molecule (Hb α-semisynthetic reaction). Unlike the other protease-catalyzed protein/peptide splicing reactions of fragment complementing systems, the enzymic condensation of nonassociating segments of Hb α is facilitated by the organic cosolvent induced α-helical conformation of product acting as the “molecular trap” of the splicing reaction. The segments α_24–30 and α_31–40 are the shortest complementary segments that can be spliced by V8 protease. In the present study, the chemistry of the contiguous segment (product) α_24–40 has been manipulated by engineering the amino acid replacements to the positions α²⁷ and α³¹ to delineate the structural basis of the molecular trap. The location of Glu²⁷ and Arg³¹ residues in the contiguous segment α_24–40 (as well as in other larger segments) is ideal to generate (i, i+4) side-chain carboxylate-guanidino interaction in its α-helical conformation. The amino acid residue replacement studies have confirmed that the side chains at α²⁷ and α³¹ facilitate the semisynthetic reaction. The relative influence of the substitute at these sites on the splicing reaction depends on the chemical nature of the side chain and the location. The γ-carboxylate guanidino side-chain interaction appears to contribute up to a maximum of 85% of the thermodynamic stability of the molecular trap. The studies also demonstrate that the thermodynamic stability of the molecular trap is determined by two interdependent conformational aspects of the peptide. One is an amino acid-sequence-specific event that facilitates the induction of an α-helical conformation to the contiguous segment in the presence of organic cosolvent that imparts some amount of protease resistance to Glu³⁰-Arg³¹ peptide bond. The second structural aspect is a site-specific event, ani, i+4 side-chain interaction in the α-helical conformation of the peptide which imparts an additional thermodynamic stability to the molecular trap. The results suggest that conformationally driven “molecular traps” of protease-mediated ligation reactions of peptides could be designed into products to facilitate the modular assembly of peptides/proteins.     

Microwave exposure induces Hsp70 and confers protection against hypoxia in chick embryos

To determine if microwave exposure could elicit a biological effect in the absence of thermal stress, studies were designed in which chick embryos were exposed to athermal microwave radiation (915 MHz) to look for induction of Hsp70, a protein produced during times of cellular stress that aids in the protection of cellular components. Levels of Hsp70 were found to increase within 2 h, with maximum expression ( approximately 30% higher than controls) typically occurring by 3 h from the start of exposure. Other embryos were exposed to microwave radiation prior to being subjected to hypoxic stress, and were found to have significantly higher survival (P < 0.05) following re-oxygenation than non-exposed controls. The results of these studies indicate that not only can athermal microwave exposures activate the stress protein response pathway; they can also enhance survivability following exposure to a subsequent, potentially lethal stress. From a public health standpoint, it is important that more studies be performed to determine if repeated exposures, a condition likely to be found in cell phone use, are still beneficial.     

Chemistry of the “molecular trap” of protease-catalyzed splicing reaction of complementary segments of α-subunit of hemoglobin A

The complementary fragments of human Hb α, α_1–30, and α_31–141 are spliced together by V8 protease in the presence of 30%n-propanol to generate the full-length molecule (Hb α-semisynthetic reaction). Unlike the other protease-catalyzed protein/peptide splicing reactions of fragment complementing systems, the enzymic condensation of nonassociating segments of Hb α is facilitated by the organic cosolvent induced α-helical conformation of product acting as the “molecular trap” of the splicing reaction. The segments α_24–30 and α_31–40 are the shortest complementary segments that can be spliced by V8 protease. In the present study, the chemistry of the contiguous segment (product) α_24–40 has been manipulated by engineering the amino acid replacements to the positions α²⁷ and α³¹ to delineate the structural basis of the molecular trap. The location of Glu²⁷ and Arg³¹ residues in the contiguous segment α_24–40 (as well as in other larger segments) is ideal to generate (i, i+4) side-chain carboxylate-guanidino interaction in its α-helical conformation. The amino acid residue replacement studies have confirmed that the side chains at α²⁷ and α³¹ facilitate the semisynthetic reaction. The relative influence of the substitute at these sites on the splicing reaction depends on the chemical nature of the side chain and the location. The γ-carboxylate guanidino side-chain interaction appears to contribute up to a maximum of 85% of the thermodynamic stability of the molecular trap. The studies also demonstrate that the thermodynamic stability of the molecular trap is determined by two interdependent conformational aspects of the peptide. One is an amino acid-sequence-specific event that facilitates the induction of an α-helical conformation to the contiguous segment in the presence of organic cosolvent that imparts some amount of protease resistance to Glu³⁰-Arg³¹ peptide bond. The second structural aspect is a site-specific event, ani, i+4 side-chain interaction in the α-helical conformation of the peptide which imparts an additional thermodynamic stability to the molecular trap. The results suggest that conformationally driven “molecular traps” of protease-mediated ligation reactions of peptides could be designed into products to facilitate the modular assembly of peptides/proteins.     

An examination of the binding behavior of histidine-containing peptides with immobilized metal complexes derived from the macrocyclic ligand, 1,4,7-triazacyclononane

In this study, two different experimental approaches have been employed to examine the binding behavior of histidine-containing peptides with metal ion complexes derived from the macrocyclic ligand 1,4,7-triazacyclononane (tacn). Firstly, a molecular modeling approach has been employed to derive the strain energies for test peptide sequences that have a predicted propensity to readily adopt an α-helical conformation. To this end, binuclear metal complexes were examined with peptides containing two histidine residues in different locations in a pair of peptides of the same composition but different sequence. These modeling results indicate that there are no energetic constraints for two-point binding to occur with dicopper(II) binuclear complexes when two histidine residues are appropriately placed in an α-helical conformation. Secondly, binding experiments were carried out to establish the effect of one or more histidine residues within a peptide sequence on the affinity of a peptide for these Cu(II)–tacn derived binuclear complexes when immobilized onto a chromatographic support material. The results confirm that for all chelating systems, higher affinity is achieved as the histidine number in the peptide structure increases, although the relative location of the histidine residues in these small peptides did not introduce a significant constraint to the conformation on interacting with the immobilized Cu(II) binuclear complexes.     

Thermotropic lipid phase separations in human erythrocyte ghosts and cholesterol-enriched rat liver plasma membranes

Summary Electron spin resonance (ESR) studies of human erythrocyte ghosts labeled with 5-nitroxide stearate, I(12,3), indicate that a temperature-dependent lipid phase separation occurs with a high onset at 38°C. Cooling below 38°C induces I(12,3) clustering. Similar phase separations were previously identified in human platelet and cholesterol-loaded [cholesterol/phospholipid molar ratio (C/P)=0.85] rat liver plasma membranes [L.M. Gordon et al., 1983;J. Membrane Biol. 76; 139–149]; these were attributed to redistribution of endogenous lipid components such that I(12,3) is excluded from cholesterol-rich domains and tends to reside in cholesterol-poor domains. Further enrichment of rat liver plasma membranes to C/P ratios of 0.94–0.98 creates an artificial system equivalent to human erythrocyte ghosts (C/P=0.90), using such criteria as probe flexibility, temperature dependent I(12,3) clustering; and polarity of the probe environment. Consequently, cholesterol-rich and-poor domains probably exist in both erythrocyte ghosts and high cholesterol liver membranes at physiologic temperatures. The temperature dependence of cold-induced hypertonic lysis of intact human erythrocytes was examined by incubating cells in 0.9m sucrose for 10 min at 1°C intervals between 9 and 46°C (Stage 1), and then subjecting them to 0°C for 10 min (Stage 2). Plots of released hemoglobin are approx. sigmoidal, with no lysis below 18°C and maximal lysis above 40°C. The protective effect of low temperatures during Stage 1 may be due to the formation of cholesterol-rich domains that alter the bilayer distribution and/or conformation of critical membrane-associated proteins.     

Microwave radiation (2.45 GHz)-induced oxidative stress: Whole-body exposure effect on histopathology of Wistar rats

Man-made microwave and radiofrequency (RF) radiation technologies have been steadily increasing with the growing demand of electronic appliances such as microwave oven and cell phones. These appliances affect biological systems by increasing free radicals, thus leading to oxidative damage. The aim of this study was to explore the effect of 2.45 GHz microwave radiation on histology and the level of lipid peroxide (LPO) in Wistar rats. Sixty-day-old male Wistar rats with 180 ± 10 g body weight were used for this study. Animals were divided into two groups: sham exposed (control) and microwave exposed. These animals were exposed for 2 h a day for 35 d to 2.45 GHz microwave radiation (power density, 0.2 mW/cm²). The whole-body specific absorption rate (SAR) was estimated to be 0.14 W/kg. After completion of the exposure period, rats were sacrificed, and brain, liver, kidney, testis and spleen were stored/preserved for determination of LPO and histological parameters. Significantly high level of LPO was observed in the liver (p < 0.001), brain (p < 0.004) and spleen (p < 0.006) in samples from rats exposed to microwave radiation. Also histological changes were observed in the brain, liver, testis, kidney and spleen after whole-body microwave exposure, compared to the control group.

Based on the results obtained in this study, we conclude that exposure to microwave radiation 2 h a day for 35 d can potentially cause histopathology and oxidative changes in Wistar rats. These results indicate possible implications of such exposure on human health.     

Biomembranes from slaughterhouse blood erythrocytes as prolonged release systems for dexamethasone sodium phosphate

The present study investigated preparation of bovine and porcine erythrocyte membranes from slaughterhouse blood as bio‐derived materials for delivery of dexamethasone‐sodium phosphate (DexP). The obtained biomembranes, i.e., ghosts were characterized in vitro in terms of morphological properties, loading parameters, and release behavior. For the last two, an UHPLC/–HESI–MS/MS based analytical procedure for absolute drug identification and quantification was developed. The results revealed that loading of DexP into both type of ghosts was directly proportional to the increase of drug concentration in the incubation medium, while incubation at 37°C had statistically significant effect on loaded amount of DexP (P < 0.05). The encapsulation efficiency was about fivefold higher in porcine compared to bovine ghosts. Insight into ghosts’ surface morphology by field emission‐scanning electron microscopy and atomic force microscopy confirmed that besides inevitable effects of osmosis, DexP inclusion itself had no observable additional effect on the morphology of the ghosts carriers. DexP release profiles were dependent on erythrocyte ghost type and amount of residual hemoglobin. However, sustained DexP release was achieved and shown over 3 days from porcine ghosts and 5 days from bovine erythrocyte ghosts. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1046–1055, 2016     

Microwave assisted Wolff rearrangement: A facile method for the synthesis of Fmoc-β-amino acids

Summary The Wolff rearrangement of α-diazoketones, derived from Fmoc-α-amino acids, under no base conditions on exposure to microwave irradiation for 40 to 60 sec to Fmoc-β-amino acids with retention of configuration in good yield (91–95%) is described.     

Cα-Methyl, Cα-phenylglycine peptides: A structural study

Summary In order to obtain further information on the role played by phenyl ring position in the C^α-methylated α-amino acid side chain on peptide preferred conformation, the crystal-state structural preferences of C^α-methyl, C^α-phenylglycine peptides have been determined by X-ray diffraction. This study shows that either the fully extended conformation or the β-bend/3₁₀-helical structures are adopted by peptides characterized by this C^α-methylated, β-branched, aromatic α-amino acid.     

Human Antimicrobial Peptides: Defensins, Cathelicidins and Histatins

Antimicrobial peptides, which have been isolated from many bacteria, fungi, plants, invertebrates and vertebrates, are an important component of the natural defenses of most living organisms. The isolated peptides are very heterogeneous in length, sequence and structure, but most of them are small, cationic and amphipathic. These peptides exhibit broad-spectrum activity against Gram-positive and Gram-negative bacteria, yeasts, fungi and enveloped viruses. A wide variety of human proteins and peptides also have antimicrobial activity and play important roles in innate immunity. In this review we discuss three important groups of human antimicrobial peptides. The defensins are cationic non-glycosylated peptides containing six cysteine residues that form three intramolecular disulfide bridges, resulting in a triple-stranded β-sheet structure. In humans, two classes of defensins can be found: α-defensins and β-defensins. The defensin-related HE2 isoforms will also be discussed. The second group is the family of histatins, which are small, cationic, histidine-rich peptides present in human saliva. Histatins adopt a random coil conformation in aqueous solvents and form α-helices in non-aqueous solvents. The third group comprises only one antimicrobial peptide, the cathelicidin LL−37. This peptide is derived proteolytically from the C-terminal end of the human CAP18 protein. Just like the histatins, it adopts a largely random coil conformation in a hydrophilic environment, and forms an α-helical structure in a hydrophobic environment.     

Many gene-regulatory proteins appear to have a similar α-helical fold that binds DNA and evolved from a common precursor

Summary Amino acid and DNA sequence comparisons suggest that many sequence-specific DNA-binding proteins have in common and homologous region of about 22 amino acids. This region corresponds to two consecutive α-helices that occur in bot Cro and cI repressor proteins of bacteriophage λ and in catabolite gene activator protein ofEscherichia coli and are presumed to interact with DNA. The results obtained here suggest that this α-helical DNA-binding fold occurs in many proteins that regulate gene expression. It also appears that this DNA-binding unit evolved from a common evolutionary precursor.     

Conformational studies of peptides representing a segment of TM7 from H+-VO-ATPase in SDS micelles

The conformation of a transmembrane peptide, sMTM7, encompassing the cytoplasmic hemi-channel domain of the seventh transmembrane section of subunit a from V-ATPase from Saccharomyces cerevisiae solubilized in SDS solutions was studied by circular dichroism (CD) spectroscopy and fluorescence spectroscopy of the single tryptophan residue of this peptide. The results show that the peptide adopts an α-helical conformation or aggregated β-sheet depending on the peptide-to-SDS ratio used. The results are compared with published data about a longer version of the peptide (i.e., MTM7). It is concluded that the bulky, positively charged arginine residue located in the center of both peptides has a destabilizing effect on the helical conformation of the SDS-solubilized peptides, leading to β-sheet formation and subsequent aggregation.     

Water exchange through erythrocyte membranes: Nuclear magnetic resonance studies on resealed ghosts compared to human erythrocytes

Summary The water diffusion across human erythrocyte membrane has been studied on intact cells and resealed ghosts by a doping NMR technique. Although the water exchange time of ghosts was longer than that of erythrocytes, no significant differences in their diffusional permeability were noticed for temperatures in the range 2–43°C. Contrary to what was previously noticed in erythrocytes, no significant increase in the water exchange time of ghosts in the acid range of pH occurred.     

Pathway of Oxidative Folding of a 3-Disulfide α-Lactalbumin May Resemble Either BPTI Model or Hirudin Model

Pathways of oxidative folding of disulfide proteins display a high degree of diversity and vary among two extreme models. The BPTI model is defined by limited species of folding intermediates adopting mainly native disulfide bonds. The hirudin model is characterized by highly heterogeneous folding intermediates containing mostly non-native disulfide bonds. αLA-IIIA is a 3-disulfide variant of α-lactalbumin (αLA) with a 3-D conformation essentially identical to that of intact αLA. αLA-IIIA contains 3 native disulfide bonds of αLA, two of them are located at the calcium binding β-subdomain (Cys⁶¹–Cys⁷⁷ and Cys⁷³–Cys⁹¹) and the third bridge is located within the α-helical domain of the molecule (Cys²⁸–Cys¹¹¹). We investigate here the pathway of oxidative folding of fully reduced αLA-IIIA with and without stabilization of its β-subdomain by calcium binding. In the absence of calcium, the folding pathway of αLA-IIIA was shown to resemble that of hirudin model. Upon stabilization of β-sheet domain by calcium binding, the folding pathway of αLA-IIIA exhibits a striking similarity to that of BPTI model. Three predominant folding intermediates of αLA-IIIA containing exclusively native disulfide bonds were isolated and structurally characterized. Our results further demonstrate that stabilization of subdomains in a protein may dictate its folding pathway and represent a major cause for the existing diversity in the folding pathways of the disulfide-containing proteins.