Identification of tubby and tubby-like protein 1 as eat-me signals by phage display

Phagocytosis is an important process for the removal of apoptotic cells or cellular debris. Eat-me signals control the initiation of phagocytosis and hold the key for in-depth understanding of its molecular mechanisms. However, because of difficulties to identify unknown eat-me signals, only a limited number of them have been identified and characterized. Using a newly developed functional cloning strategy of open reading frame (ORF) phage display, we identified nine putative eat-me signals, including tubby-like protein 1 (Tulp1). This further led to the elucidation of tubby as the second eat-me signal in the same protein family. Both proteins stimulated phagocytosis of retinal pigment epithelium (RPE) cells and macrophages. Tubby-conjugated fluorescent microbeads facilitated RPE phagocytosis. Tubby and Tulp1, but not other family members, enhanced the uptake of membrane vesicles by RPE cells in synergy. Retinal membrane vesicles of Tubby mice and Tulp1^−/− mice showed reduced activities for RPE phagocytosis, which were compensated by purified tubby and Tulp1, respectively. These data reveal a novel activity of tubby and Tulp1, and demonstrate that unbiased identification of eat-me signals by the broadly applicable strategy of ORF phage display can provide detailed insights into phagocyte biology.     

A fluorimetric semi-microplate format assay of protein carbonyls in blood plasma

Oxidative stress, originating from reactive oxygen species (ROS), has been implicated in aging and various human diseases. The ROS generated can oxidize proteins producing protein carbonyl derivatives. The level of protein carbonyls in blood plasma has been used as a measure of overall oxidative stress in the body. Classically, protein carbonyls have been quantitated spectrophotometrically by directly reacting them with 2,4-dinitrophenylhydrazine (DNPH). However, the applicability of this method to biological samples is limited by its low inherent sensitivity. This limitation has been overcome by the development of sensitive enzyme-linked immunosorbent assay (ELISA) methods to measure protein carbonyls. As part of the Healthy Aging in Neighborhoods of Diversity across the Lifespan (HANDL) study, oxidative stress in humans was quantified by measuring blood plasma protein carbonyls using the two commercially available ELISA kits and the spectrophotometric DNPH assay. Surprisingly, two ELISA methods gave very different values for protein carbonyls, both of which were different from the value of the spectrophotometric method. We have developed a fluorescent semi-microplate format assay of protein carbonyls involving direct reaction of protein carbonyls with fluorescein thiosemicarbazide that correlates (R = 0.992) with the direct spectrophotometric method. It has a coefficient of variation of 4.99% and is at least 100 times more sensitive than the spectrophotometric method.     

Pharmacology of mitochondrial potassium channels: dark side of the field

Mitochondrial potassium channels play an important role in cytoprotection. Potassium channels in the inner mitochondrial membrane are modulated by inhibitors and activators (potassium channel openers) previously described for plasma membrane potassium channels. The majority of mitochondrial potassium channel modulators exhibit a broad spectrum of off-target effects. These include uncoupling properties, inhibition of the respiratory chain and effects on cellular calcium homeostasis. Therefore, the rational application of channel inhibitors or activators is crucial to understanding the cellular consequences of mitochondrial channel inhibition or activation. Moreover, understanding their side-effects should facilitate the design of a specific mitochondrial channel opener with cytoprotective properties. In this review, we discuss the complex interactions of potassium channel inhibitors and activators with cellular structures.     

Screening the Fusarium graminearum inhibitory mutant strain from Bacillus subtilis by atmospheric-pressure plasma jet

Aims:  The aim of this study was to improve the antagonistic activity of Bacillus subtilis JA towards Fusarium graminearum by screening high-yielding mutant using the atmospheric-pressure plasma jet (APPJ).
Methods and Results:  Atmospheric-pressure plasma jet was applied as mutagenic source for breeding high-yielding mutant strain. Helium was used as APPJ operating gas. The mutation effects of different treatment times of APPJ were studied. The mutant strain designated as B. subtilis B06 was successfully screened out, which showed higher antagonistic activity against F. graminearum in vitro . Its inhibition zone against the indicator fungus increased by 23% compared to the original one. HPLC and ESI (electrospray ionization) mass spectrometry analysis indicated that antifungal compounds produced by the mutant and original strain belonged to the lipopeptide, surfactin and iturin families. The mutant strain showed favourable properties of faster growth in the fermentation process and higher production of antibiotics. The lipopeptide production of the mutant was 2·3-fold as that of the original strain.
Conclusions:  A mutant strain with strong antagonistic activity and high yielding of antibiotics was obtained by APPJ in this study. The mutant could be used as a promising biocontrol agent in agriculture.
Significance and Impact of Study:  This study provides a novel mutagenic source for breeding high-yielding microbial mutant, which would be very useful in the application of some valuable metabolites from micro-organism.     

Biological responses of Bacillus stratosphericus to Floating Electrode‐Dielectric Barrier Discharge Plasma Treatment

Aims: Dielectric barrier discharge (DBD) plasma is used for sterilization of contaminated inanimate surfaces but seldomly optimized and depends upon the type of organisms and the plasma treatment duration, (net energy deposited) this efficacy varies. The proposed study was designed to see biological responses of one of the robust organism, Bacillus stratosphericus. Methods and Results: DBD plasma was applied over various durations to B. stratosphericus either surface‐dried or suspension in de‐ionized water, and viability, culturability, and viable but nonculturability (VBNC) were assayed using standard techniques. Depending upon the exposure of B. stratosphericus to DBD plasma resulted in three viability states, viable and culturable at low plasma doses and VBNC or disintegrated bacteria at higher plasma doses. Although organism’s respiration levels at relatively low levels, immediately after plasma treatment, over the course of 24‐ h respiratory activity was increased c. eight times (and found still nonculturable during colony assays). Conclusions: The loss of culturability is hypothesized to be induced as one of the responses to oxidative stress and it remains to be unclear if the response is temporary or indefinite. Appropriate plasma powers should be used to avoid VBNC‐like status. 2,3‐Bis‐(2‐methoxy‐4‐nitro‐5‐sulfophenyl)‐2H‐tetrazolium‐5‐carboxanilide (XTT) assay is a good alternative method to detect VBNC state. Significance and Impact of the Study: Bacillus stratosphericus has the potential to turn into VBNC upon plasma application, and XTT assay can be an alternative method to detect VBNC state.     

Non-stereo-selective cytosolic human brain tissue 3-ketosteroid reductase is refractory to inhibition by AKR1C inhibitors

Cerebral 3α-hydroxysteroid dehydrogenase (3α-HSD) activity was suggested to be responsible for the local directed formation of neuroactive 5α,3α-tetrahydrosteroids (5α,3α-THSs) from 5α-dihydrosteroids. We show for the first time that within human brain tissue 5α-dihydroprogesterone and 5α-dihydrotestosterone are converted via non-stereo-selective 3-ketosteroid reductase activity to produce the respective 5α,3α-THSs and 5α,3β-THSs. Apart from this, we prove that within the human temporal lobe and limbic system cytochrome P450c17 and 3β-HSD/Δ^5–4 ketosteroid isomerase are not expressed. Thus, it appears that these brain regions are unable to conduct de novo biosynthesis of Δ⁴-3-ketosteroids from Δ⁵-3β-hydroxysteroids. Consequently, the local formation of THSs will depend on the uptake of circulating Δ⁴-3-ketosteroids such as progesterone and testosterone. 3α- and 3β-HSD activity were (i) equally enriched in the cytosol, (ii) showed equal distribution between cerebral neocortex and subcortical white matter without sex- or age-dependency, (iii) demonstrated a strong and significant positive correlation when comparing 46 different specimens and (iv) exhibited similar sensitivities to different inhibitors of enzyme activity. These findings led to the assumption that cerebral 3-ketosteroid reductase activity might be catalyzed by a single enzyme and is possibly attributed to the expression of a soluble AKR1C aldo-keto reductase. AKR1Cs are known to act as non-stereo-selective 3-ketosteroid reductases; low AKR1C mRNA expression was detected. However, the cerebral 3-ketosteroid reductase was clearly refractory to inhibition by AKR1C inhibitors indicating the expression of a currently unidentified enzyme. Its lack of stereo-selectivity is of physiological significance, since only 5α,3α-THSs enhance the effect of GABA on the GABA_A receptor, whereas 5α,3β-THSs are antagonists.     

Women's intercollegiate volleyball and tennis: Effects of warm-up, competition, and practice on saliva levels of cortisol and testosterone

In virtually all sports, participants “warm-up” prior to formal competition. Women athletes from a highly ranked varsity college volleyball team and, in a second study, a highly ranked varsity college tennis team gave saliva samples before warm-up, at mid-warm-up (volleyball) or after warm-up (tennis), and immediately after intercollegiate competition. For volleyball and tennis, warm-up was associated with a substantial elevation in saliva levels of testosterone which was carried over through the period of actual competition. Cortisol levels were relatively unchanged during warm-up, but typically rose during competition. Thus, as women prepare for athletic competition by warming up, testosterone levels rise in apparent anticipation of the coming contest and then remain high through the period of play. In volleyball and tennis, after-practice testosterone level was significantly higher than before-practice level, and practice session increases in testosterone (but not cortisol) were positively correlated with increases in testosterone during intercollegiate competition. When practice and competitive play share as yet undetermined key elements, individual differences in this endocrine response to “competition” appear stable across practice and intercollegiate competition.     

A HPLC-UV method for the determination of puerarin in rat plasma after intravenous administration of PEGylated puerarin conjugate

A sensitive and reproducible HPLC method for quantitative determination of puerarin (PUE) in rat plasma was developed and validated using 4-hydroxybenzaldehyde as an internal standard. The separation of PUE was performed on a CAPCELL PAK C18 column by gradient elution with 0.2% aqueous phosphoric acid and acetonitrile as the mobile phase. The method was validated and found to be linear in the range of 80-12,000ng/mL. The limit of quantification was 80ng/mL based on 100μL of plasma. The variations for intra- and inter-day precision were less than 8.3%, and the accuracy values were between 98% and 105.2%. The extraction recoveries were more than 85%. The method was successfully applied in the comparative study of pharmacokinetics of PEGylated puerarin (PEG-PUE) versus PUE in rats. Compared with PUE, PEG-PUE showed a 5.2-fold increase in half-life of PUE and a 4.7-fold increase in mean residence time. In addition, this method was also successfully applied to determine the low plasma concentration of PUE regenerated from PEG-PUE in vitro.