Denaturation of α-lactalbumin and myoglobin by the anionic biosurfactant rhamnolipid

Glycolipid biosurfactants (GBS) are promising environmentally friendly alternatives to chemical surfactants. Surfactants interact with proteins in many applications, often leading to significant changes in protein properties. Given GBS' marked difference in structure compared to traditional chemical surfactants, it is of interest to investigate their impact on protein structure and stability. Here we combine spectroscopic and calorimetric studies to analyze the interactions between the anionic GBS rhamnolipid (RL) and two model proteins α-lactalbumin in the Ca^2 +-free apo-form (αLA) and myoglobin (Mb), whose interactions with traditional surfactants are well known. RL denatures αLA at sub-cmc concentrations (0.1–1 mM) while Mb is only denatured above the cmc, i.e. in the presence of RL micelles. Denaturation leads to increased α-helicity, similar to the effect of SDS. The proteins bind approximately the same amount of RL by weight as SDS. However, RL employs a denaturation mechanism which combines features from non-ionic surfactants (very slow unfolding kinetics and few unfolding steps) with those of SDS (unfolding below the cmc in the case of αLA and the ability to unfold stable proteins in the case of Mb). We ascribe these features to RL's weakly acidic carboxylic head group and complex hydrophobic tail, which lead to a low cmc and low protein affinity. These features restrict the concentration range where RL monomers can bind and denature proteins while still allowing micelles to bind and denature to a significant extent.     

Denaturation of proteins by ascorbic acid: Effects on dopamine-β-hydroxylase

Under conditions that are optimum for DH, ascorbic acid denatures serum albumin, -globulin, catalase, and D\H. With ascorbate plus Cu²⁺, the proteins arc almost completely destroyed. Pyrazole protects DH and albumin, but not catalase. Superoxide dismutase (SOD) is not denatured by ascorbate, with or without Cu²⁺, and in combination with catalytic amounts of catalase or Fe²⁺ it stimulates maximum DH activity. In other words, a combination of catalase and SOD, or Fe²⁺ and SOD, will protect DH. Excessive amounts of catalase and/or other protein, either native or denatured will prevent the effects of superoxide and/or ascorbate, but cannot replace the requirements for catalytic quantities of catalase or Fe²⁺. The results suggest that the rate of hydroxylation of tyramine may be limited by superoxide, but that the latter per se does not denature DH as does hydrogen peroxide. The in vitro activation of oxygen by DH is a toxic process, involving the production of both hydrogen peroxide and superoxide and possibly other free radicals. In the absence of precise regulation of the production and concentrations of these compounds, the enzyme is denatured.     

Immunogenicity of Rabies Virus Inactivated by β-Propiolactone, Acetylethyleneimine, and Ionizing Irradiation

Ionizing radiation, beta-propiolactone, and acetylethyleneimine were compared for their ability as virus-inactivating agents for the preparation of rabies vaccine. Each agent reduced viral infectivity exponentially; ionizing radiation also destroyed viral hemagglutinin. The vaccine prepared by ionizing radiation was equal or superior to that prepared by beta-propiolactone in its ability to protect mice from rabies infection. The acetylethyleneimine-treated vaccine was a less potent immunogen.     

Reversible heme-dependent regulation of human cystathionine β-synthase by a flavoprotein oxidoreductase

Human CBS is a PLP-dependent enzyme that clears homocysteine, gates the flow of sulfur into glutathione, and contributes to the biogenesis of H(2)S. The presence of a heme cofactor in CBS is enigmatic, and its conversion from the ferric- to ferrous-CO state inhibits enzyme activity. The low heme redox potential (-350 mV) has raised questions about the feasibility of the ferrous-CO state forming under physiological conditions. Herein, we provide the first evidence of reversible inhibition of CBS by CO in the presence of a human flavoprotein and NADPH. These data provide a mechanism for cross talk between two gas-signaling systems, CO and H(2)S, via heme-mediated allosteric regulation of CBS.     

Enhanced Photocatalytic Performance of ZnS for Reversible Amination of α-oxo Acids by Hydrothermal Treatment

To understand how life could have originated on early Earth, it is essential to know what biomolecules and metabolic pathways are shared by extant organisms and what organic compounds and their chemical reaction channels were likely to have been primordially available during the initial phase of the formation of prebiotic metabolism. In a previous study, we demonstrated for the first time the reversible amination of α-oxo acids on the surface of photo-illuminated ZnS. The sulfide mineral is a typical component at the periphery of submarine hydrothermal vents which has been frequently argued as a very attractive venue for the origin of life. In this work, in order to simulate more closely the precipitation environments of ZnS in the vent systems, we treated newly-precipitated ZnS with hydrothermal conditions and found that its photocatalytic power was significantly enhanced because the relative crystallinity of the treated sample was markedly increased with increasing temperature. Since the reported experimental conditions are believed to have been prevalent in shallow-water hydrothermal vents of early Earth and the reversible amination of α-oxo acids is a key metabolic pathway in all extant life forms, the results of this work provide a prototypical model of the prebiotic amino acid redox metabolism. The amino acid dehydrogenase-like chemistry on photo-irradiated ZnS surfaces may advance our understanding of the establishment of archaic non-enzymatic metabolic systems.     

Differential Scanning Calorimetrie Study of the Thermal Denaturation of Almond β-Glucosidase

The thermal denaturation of almond β-glucosidase [EC 3.2.1.21] was studied by differential scanning calorimetry. The shape of the DSC trace was highly dependent on pH; two peaks were observed between pH 6–8, but only one peak between pH 4–5. All of the DSC curves were resolved into three components according to the model of independent two-state processes, and the thermodynamic parameters for the denaturation were evaluated. The dependence of the shape of DSC curves was accounted for mainly by the rapid changes of denaturation enthalpy and denaturation temperature of the third component in the acidic pH region.     

Comparison of the Regulation of β-Catenin Signaling by Type I,Type II and Type III Interferons in Hepatocellular Carcinoma Cells

Background/Objective

IFNs are a group of cytokines that possess potent antiviral and antitumor activities, while β-catenin pathway is a proliferative pathway involved in carcinogenesis. Interaction between these two pathways has not been well elaborated in hepatocellular carcinoma (HCC).

Methods

HCC cell lines, HepG2 and Huh7, were used in this study. β-catenin protein levels and corresponding signaling activities were observed by flow cytometry and luciferase assay, respectively. Cell proliferation was quantified by counting viable cells under microscope, and apoptosis by TUNEL assay. DKK1 and GSK3β levels were determined by flow cytometry. Secreted DKK1 was tested by ELISA. FLUD, S3I and aDKK1 were used to inhibit STAT1, STAT3 and DKK1 activities, respectively.

Results

Our findings show that all three types of IFNs, IFNα, IFNγ and IFNλ, are capable of inhibiting β-catenin signaling activity in HepG2 and Huh7 cells, where IFNγ was the strongest (p<0.05). They expressed suppression of cellular proliferation and induced apoptosis. IFNγ expressed greater induction ability when compared to IFNα and IFNλ (p<0.05). All tested IFNs could induce DKK1 activation but not GSK3β in HepG2 and Huh7 cells. IFNs induced STAT1 and STAT3 activation but by using specific inhibitors, we found that only STAT3 is vital for IFN-induced DKK1 activation and apoptosis. In addition, DKK1 inhibitor blocked IFN-induced apoptosis. The pattern of STAT3 activation by different IFNs is found consistent with the levels of apoptosis with the corresponding IFNs (p<0.05).

Conclusions

In hepatocellular carcinoma, all three types of IFNs are found to induce apoptosis by inhibiting β-catenin signaling pathway via a STAT3- and DKK1-dependent pathway. This finding points to a cross-talk between different IFN types and β-catenin signaling pathways which might be carrying a biological effect not only on HCC, but also on processes where the two pathways bridge.     

Reversible protection of Cys-93(β) by PEG alters the structural and functional properties of the PEGylated hemoglobin

As a potential hemoglobin (Hb)-based oxygen carrier (HBOC), the PEGylated Hb has received much attention for its non-nephrotoxicity. However, PEGylation can adversely alter the structural and functional properties of Hb. The site of PEGylation is an important factor to determine the structure and function of the PEGylated Hb. Thus, protection of some sensitive residues of Hb from PEGylation is of great significance to develop the PEGylated Hb as HBOC. Here, Cys-93(β) of Hb was conjugated with 20 kDa polyethylene glycol (PEG20K) through hydrazone and disulfide bonds. Then, the conjugate was modified with PEG5K succinimidyl carbonate (PEG5K-SC) using acylation chemistry, followed by removal of PEG20K Hb with hydrazone hydrolysis and disulfide reduction. Reversible conjugation of PEG20K at Cys-93(β) can protect Lys-95(β), Val-1(α) and Lys-16(α) of Hb from PEGylation with PEG5K-SC. The autoxidation rate, oxygen affinity, structural perturbation and tetramer instability of the PEGylated Hb were significantly decreased upon protection with PEG20K. The present study is expected to improve the efficacy of the PEGylated Hb as an oxygen therapeutic.     

“Pollination” of a fungus by a fly

Summary The mechanism resulting in fertilization of Epichloë typhina, a heterothallic ascomycete that is an endophytic pathogen of grasses, has now been discovered. Conidia of one mating type are produced in stromata and are then transferred by insects to individuals of the opposite mating type. One insect, Phorbia phrenione, is a particularly important vector of conidia. Once conidia of the opposite mating type have been transferred to a stroma, the life cycle continues with the formation of perithecia.     

Isolation of protoplasts by means of a “species-specific” autolysine inChlamydomonas

Summary Prior to fusion, gametes ofChlamydomonas reinhardii discard their cell walls. This naturally occuring phenomenon has provided the basis for a method of protoplast isolation from both gametes and vegetative cells within the genusChlamydomonas. When synchronized cultures of compatibleChlamydomonas gametes are mixed it is possible, after removal of the cells, to obtain a solution having a high cell wall lytic activity. That vegetative and gamete cells after treatment with this gamete-autolysine are indeed protoplasts has been proved by various light and electron microscopical methods.The species specifity of this autolysine, its difference to the previously described sporangialautolysine (Schlösser 1966) and furthermore its use in the large scale production of protoplasts is also described. Since this wall autolysine is a factor produced by the cells themselves at a particular stage in their life cycle it represents a non-foreign agent in contrast to all other enzymic methods previously employed for protoplast isolation.     

The game of “gossip” analyzed by the theory of information

This paper deals with an analysis of a distributed model of the game of “gossip”, in which a message is passed through a line of individuals, and the final (in general, garbled) result is compared with the original ungarbled message. The deterioration of information (defined in the sense of Shannon and Wiener) along the line is calculated, and exact as well as asymptotic formulas suggesting approximate linear electric network analogues are obtained.     

Blood Glucose Homeostasis in the Course of Partial Pancreatectomy – Evidence for Surgically Reversible Diabetes Induced by Cholestasis

Background and Aim

Partial pancreatic resection is accompanied not only by a reduction in the islet cell mass but also by a variety of other factors that are likely to interfere with glucose metabolism. The aim of this work was to characterize the patient dynamics of blood glucose homeostasis during the course of partial pancreatic resection and to specify the associated clinico-pathological variables.

Methods

In total, 84 individuals undergoing elective partial pancreatic resection were consecutively recruited into this observational trial. The individuals were assigned based on their fasting glucose or oral glucose tolerance testing results into one of the following groups: (I) deteriorated, (II) stable or (III) improved glucose homeostasis three months after surgery. Co-variables associated with blood glucose dynamics were identified.

Results

Of the 84 participants, 25 (30%) displayed a normal oGTT, 17 (20%) showed impaired glucose tolerance, and 10 (12%) exhibited pathological glucose tolerance. Elevated fasting glucose was present in 32 (38%) individuals before partial pancreatic resection. Three months after partial pancreatic resection, 14 (17%) patients deteriorated, 16 (19%) improved, and 54 (64%) retained stable glucose homeostasis.Stability and improvement was associated with tumor resection and postoperative normalization of recently diagnosed glucose dysregulation, preoperatively elevated tumor markers and markers for common bile duct obstruction, acute pancreatitis and liver cell damage. Improvement was linked to preoperatively elevated insulin resistance, which normalized after resection and was accompanied by a decrease in fasting- and glucose-stimulated insulin secretion.

Conclusions

Surgically reversible blood glucose dysregulation diagnosed concomitantly with a (peri-) pancreatic tumor appears secondary to compromised liver function due to tumor compression of the common bile duct and the subsequent increase in insulin resistance. It can be categorized as “cholestasis-induced diabetes” and thereby distinguished from other forms of hyperglycemic disorders.     

Reversible removal of SO2 at low temperature by Bacillus licheniformis immobilized on γ-Al2O3

Bacillus licheniformis R08 biomass was immobilized on γ-Al₂O₃ and the effects of R08 biomass loading, SO₂ concentration, water vapor, oxygen and temperature on removal of SO₂ were investigated. The experimental results indicated that SO₂ saturation capacity increased with increasing R08 biomass loading and SO₂ concentration, but decreased with increasing adsorption temperature. Water vapor activated the adsorbent and promoted SO₂ removal. An increase in oxygen concentration from 5 to 10% had little effect on SO₂ removal. FTIR analysis revealed that the R08 biomass bound to γ-Al₂O₃ mainly by forming R-CO-O-Al bonds. X-ray photoelectron spectroscopy analysis indicated that γ-Al₂O₃ reacted with SO₂ and formed aluminum sulfate in the presence of oxygen when R08 biomass loading was 13.8%, but that amido groups of the R08 biomass reacted with SO₂ and formed sulfite when biomass loading was 32.4%. Ten continuous adsorption-desorption cycles showed that the adsorbent had an excellent regeneration performance.     

Reversible Plastidenumwandlung bei der Mutante “Weissherz” vonOenothera suaveolens Desf

Ohne ZusammenfassungMit 1 Textabbildung     

Electron-microscope cytochemistry of acetylcholine-like cation by means of low-temperature “ionic fixation”

Summary A fresh preparation of frog neuromuscle was fixed at low temperatures (0°–4°C) by means of an ionic-fixation procedure which is based on the precipitation of quaternary ammonium cations, such as choline and acetylcholine, with molybdic or tungstic heteropolyanions. A low temperature was used to slow down drastically the biological processus of vesicular exocytosis so that ionic fixation, the speed of which is only slightly influenced by temperature variation, could be performed efficiently. In addition to the conventional point-like precipitate in the synaptic vesicle which is considered to be vesicular acetylcholine, numerous spot-like precipitates were observed in the synaptic cleft. Most of these were contiguous to the active zone, and some were in a paired form and corresponded to the double rows of the synaptic vesicles in contact with active zones. It is concluded that these spot-like precipitates were acetylcholine-like cations of the synaptic vesicles which had been discharged into the synaptic cleft by exocytosis and captured by the ionic fixation procedure. The results are discussed in relation to the vesicular and non-vesicular hypothesis of acetylcholine release.