Bidirectional effect of met-enkephalin on macrophage effector functions

Summary Met-enkephalin (ME) exerts a bimodal effect on functional activities of rat peritoneal macrophages (PM); in a range of low concentration (10^-9-10^-7 M) antibody dependent cellular cytotoxicity (ADCC)was markedly stimulated with a simultaneous decrease of Fc receptor (FcR) mediated phagocytosis while the opposite was observed at 10^-6-10^-5 M concentrations.Studying the possible underlying mechanism(s) the followings were recorded: (1) ME in all applied concentrations induced an early Na⁺ influx which was followed by a Ca²⁺ efflux in the range of low concentrations. In the range of high concentrations Na⁺ influx was accompanied by a Ca²⁺ influx. (2) ME at 10^-8 M concentration induced a rise in cGMP level with a plateau in the 60–120th min of incubation. This effect was prevented by 10^-5 M of naloxone. At 10^-6 M concentration a transient rise of cAMP level was recorded which was not affected by naloxone. (3) Verapamil in 10^-6 M abolished both the Ca²⁺ influx and the rise in cAMP level induced by 10^-6-10^-5 M ME but not the rise in cGMP level induced by lower ME concentrations. (4) cAMP elevation by high ME concentrations was abolished by enkephalinase inhibitory puromycin. (5) PM-enkephalinase as assessed by the cleavage of fluorogenic substrate L-alanine beta naphthylamide (ABNA), was inhibited by 10^-6-10^-5 M of ME. This inhibition was abolished by verapamil, but not affected by naloxone. In the range of low concentrations ME appears to act on specific delta opioid receptors and its action is positively coupled to guanylate cyclase. In relatively higher concentrations ME-action is not mediated by specific delta opioid receptors and it appears to involve Ca²⁺ influx, adenylate cyclase activation as well as the processing of hormone by PM-enkephalinase.     

Light requirement and photosynthetic cell cultivation – Development of processes for efficient light utilization in photobioreactors

Although the potential of photosyntheticmicroorganisms for production of various metabolitesand in environmental bioremediation is recognized,their practical application has been limited by thedifficulty in supplying light efficiently tophotobioreactors. Various types of photobioreactorwith high illumination to volume ratios have beenproposed, but most are limited by cost, mass transfer,contamination, scale-up or a combination of these.The problem of light supply to photobioreactorscan be solved by developing photosynthetic cellcultivation systems where light is either substitutedor supplemented. Many strains of photosynthetic cellsare capable of heterotrophic growth under darkconditions and their heterotrophic culture can be usedfor efficient production of biomass and somemetabolites. However, light is absolutely required forefficient production of some metabolites. In suchcases, there is a need to supplement the heterotrophicwith photoautotrophic metabolism. Inphotoheterotrophic (mixotrophic) culture, thephotoautotrophic and heterotrophic metabolisms can beexploited for efficient production of usefulmetabolites but it has many problems such as processoptimization in terms of making a balance between thephotoautotrophic and heterotrophic metabolism. Another promising system is the sequentialheterotrophic/ photoautotrophic cultivation system,where the cells are cultivated heterotrophically tohigh concentrations and then passed through aphotobioreactor for accumulation of the desiredmetabolite(s). Furthermore, cyclicphotoautotrophic/heterotrophic cultivation system canbe used to achieve continuous cell growth underday/night cycles. This involves cultivating thecells photoautotrophically using solar light duringthe day and then adding controlled amount of organiccarbon source during the night for heterotrophicgrowth. In this review, these various systems arediscussed with some specific examples.     

Study on the interaction between anti‐tuberculosis drug ethambutol and bovine serum albumin: multispectroscopic and cyclic voltammetric approaches

The binding of bovine serum albumin (BSA) to ethambutol (EMB) was investigated using spectroscopic methods, viz., fluorescence, Fourier transform infrared (FTIR), ultraviolet (UV)/vis absorption and cyclic voltammetry techniques. Spectroscopic analysis of the emission quenching at different temperatures revealed that the quenching mechanism of serum albumin by EMB is static, which was also confirmed by lifetime measurements. The number of binding sites, n, and binding constant, K, were obtained at various temperatures. The distance, r, between EMB and the protein was evaluated according to the Förster energy transfer theory. Based on displacement experiments using site probes, viz., warfarin, ibuprofen and digitoxin, the site of binding of EMB in BSA was proposed to be Sudlow's site I. The effect of EMB on the conformation of BSA was analyzed by using synchronous fluorescence spectra (SFS) and 3D fluorescence spectra. The results of fluorescence, UV/vis absorption and FTIR spectra showed that the conformation of BSA was changed in the presence of EMB. The thermodynamic parameters including enthalpy change (ΔH⁰), entropy change (ΔS⁰) and free energy change (ΔG⁰) for BSA–EMB were calculated according to the van't Hoff equation and are discussed.     

The role of octopamine and cyclic AMP in regulating hormone release from corpora cardiaca of the American cockroach

Incubation of corpora cardiaca from adult male Periplaneta americana in the presence of octopamine results in elevated tissue levels of cyclic AMP. The octopamine-induced elevation of cyclic AMP is partially blocked by phentolamine, gramine and cyproheptadine but not by propranolol. Dopamine and 5-hydroxytryptamine also increase cyclic AMP levels in the corpus cardiacum and additivity studies indicate that separate octopamine- and dopamine-binding sites are present within the tissue. Cyclic AMP levels in the corpus cardiacum also increase in response to electrical stimulation of nervi corporis cardiaci II (NCC II) and the electrically induced effect is eliminated in the presence of phentolamine.A factor, which causes elevated haemolymph trehalose levels when injected into adult cockroaches, is released from corpora cardiaca incubated in the presence of octopamine. The active factor is denatured by incubation in the presence of pronase. The hypertrehalosemic factor is also released when corpora cardiaca are incubated in the presence of dibutyryl cyclic AMP or 40 mM potassium chloride; however dopamine and 5-hydroxytryptamine fail to effect a marked release of the hypertrehalosemic factor.The results are discussed in light of the proposal that the release of hypertrehalosemic hormone from corpora cardiaca is regulated by octopaminergic neurones contained within NCC II.     

A Novel Electrochemically Active and Fe(III)-reducing Bacterium Phylogenetically Related to Clostridium butyricum Isolated from a Microbial Fuel Cell

An obligatory anaerobic bacterium was isolated from a mediator-less microbial fuel cell using starch processing wastewater as the fuel and designated as EG3. The isolate was Gram-positive, motile and rod (2.8–3.0 μm long, 0.5–0.6 μm wide). The partial 16S rRNA gene sequence and analysis of the cellular fatty acids profile suggested that EG3 clusters with Clostridium sub-phylum and exhibited the highest similarity (98%) with Clostridium butyricum. The temperature and pH optimum for growth were 37°C and 7.0, respectively. The major products of glucose and glucose/Fe(O)OH metabolism were lactate, formate, butyrate, acetate, CO₂and H₂. Growth was faster at the initial phase and the cell yield was higher when the medium was supplemented with Fe(O)OH than without Fe(O)OH. These results suggest that Fe(III) ion is utilised as an electron sink. Cyclic voltammetry showed that Clostridium butyricum EG3 cells were electrochemically active. It is a novel characteristic of strict anaerobic Gram-positive bacteria.     

circRNA_0058097 promotes tension-induced degeneration of endplate chondrocytes by regulating HDAC4 expression through sponge adsorption of miR-365a-5p

Excessive mechanical tension can lead to the degeneration of endplate chondrocytes. The presence of tension-sensitive circRNA_0058097 molecules has been detected in human endplate chondrocytes, where it was found to be a potential competing endogenous RNA. Indeed, inhibiting the expression of circRNA_0058097 effectively enhanced the stress resistance of endplate chondrocytes, suggesting that it may be an important trigger point for the degeneration of endplate cartilage. Through a series of experiments, we reveal that circRNA_0058097 can upregulate the expression of downstream target gene histone deacetylase 4 by sponge adsorption of miR-365a-5p, which promoted morphological changes of endplate chondrocytes, and increased extracellular matrix degradation and degeneration of endplate cartilage. Therefore, circRNA_0058097 may provide a new way to prevent and treat endplate cartilage degeneration.     

Isolation of Bacillus and Paenibacillus Bacterial Strains That Produce Large Molecules of Cyclic Isomaltooligosaccharides

Cyclic isomaltooligosaccharides (CIs) usually consist of 7 to 12 glucose units, although only CI-10 has strong inclusion complex-forming ability. Four Bacillus strains and two Paenibacillus strains were isolated as novel CI-producing bacteria. Among these, five strains produced small amounts of CI-7 to CI-9, but mainly produced CI-10 to CI-12. Larger CIs, up to CI-17, were also identified.     

Spectroscopic,electrochemical and molecular docking studies of dothiepin and doxepin with bovine serum albumin and DNA base

The interaction of dothiepin (DOT) and doxepin (DOX) with bovine serum albumin (BSA) and a DNA base (adenine) was studied using UV–visible, fluorescence, attenuated total reflection–infra‐red (ATR‐IR), cyclic voltammetry and molecular docking methods. Strong fluorescence quenching was observed upon interaction of DOT and DOX with BSA/adenine and the mechanism suggested static quenching. Hydrophobic and hydrogen bonding interactions were the predominant intermolecular forces needed to stabilize the copolymer. Upon addition of the drugs: (i) the tautomeric equilibrium structure of the adenine was changed; and (ii) the oxidation and the reduction peaks of the adenine/BSA interaction shifted towards high and low potentials, respectively. In ATR‐IR, the band shift of amides I and II indicated a change in secondary structure of BSA upon binding to DOT and DOX drugs. The reduction in voltammetric current in the presence of BSA/adenine was attributed to slow diffusion of BSA/adenine binding with DOX/DOT. The docking method indicated that the drug moiety interacted with the BSA molecule. Copyright © 2016 John Wiley & Sons, Ltd.     

The mechanism of ciliary stimulation by acetylcholine: roles of calcium, PKA, and PKG

Stimulation of ciliary cells through muscarinic receptors leads to a strong biphasic enhancement of ciliary beat frequency (CBF). The main goal of this work is to delineate the chain of molecular events that lead to the enhancement of CBF induced by acetylcholine (ACh). Here we show that the Ca(2+), cGMP, and cAMP signaling pathways are intimately interconnected in the process of cholinergic ciliary stimulation. ACh induces profound time-dependent increase in cGMP and cAMP concentrations mediated by the calcium-calmodulin complex. The initial strong CBF enhancement in response to ACh is mainly governed by PKG and elevated calcium. The second phase of CBF enhancement induced by ACh, a stable moderately elevated CBF, is mainly regulated by PKA in a Ca(2+)-independent manner. Inhibition of either guanylate cyclase or of PKG partially attenuates the response to ACh of [Ca(2+)](i), but completely abolishes the response of CBF. Inhibition of PKA moderately attenuates and significantly shortens the responses to ACh of both [Ca(2+)](i) and CBF. In addition, PKA facilitates the elevation in [Ca(2+)](i) and cGMP levels induced by ACh, whereas an unimpeded PKG activity is essential for CBF enhancement mediated by either Ca(2+) or PKA.     

Inhibition of bovine α-chymotrypsin by cyclic trypsin inhibitor SFTI-1 isolated from sunflower seeds and its two acyclic analogues

Summary Trypsin inhibitor SFTI-1 isolated from sunflower seeds (comprising 14 amino acid residues and two cycles: head-to-tail cyclisation and disulfide bridge) is the smallest naturally occurring plant serine proteinase inhibitor. In our recent paper we have shown that the elimination head-to-tail cyclisation did not change trypsin inhibitory activity as judged by measured by association equilibrium constants K _a . The removal of disulfide bridge produced 2.4-fold lower activity. In the present paper we described chymotrypsin inhibitory activity. SFTI-1 inhibits significantly lower bovine α-chymortypsin (K _a =(5.20±1.56)×10⁶ M⁻¹). The activity of the analogue with disulfide bridge only was practically the same, whereas the K _a value determined for homodetic peptide was almost 3-fold lower. Considering the results obtained and the recent literature data we postulate the lower inhibitory activity against both enzymes of the analogue with head-to-tail cyclisation only reflect its lower proteolytic stability.