Kinetic study on starch hydrolysis using a glucose/maltose sensing system

Summary A dual-enzyme electrode flow injection system that can simultaneously determine glucose and maltose is used for an on-line study of starch hydrolyses catalysed by amylases. With the working system, determinations can be made every 2 minutes. A 10 L sample size with recycled back-flow minimises any loss of the reaction medium. The production, growth and decay of glucose and maltose concentrations during starch hydrolysis under various enzymatic conditions can thus be closely monitored, making it useful for the study of the catalytic kinetics of amylases and in screening and analysing enzyme systems.     

Identification of an epithelial protein related to the desmosome and intermediate filament network

Using a mAb, referred to as 08L, we have identified a protein, of M(r) approximately 140,000, associated with desmosomes of epithelial cells. The 08L antibody stained the intracellular side of lateral cell margins of monolayer epithelial cells but did not stain cell margins free of cell contact. Immunoelectron microscopy revealed that the 08L antigen was localized to the cytosolic surface of the desmosomal plaque near points of intermediate filament convergence with apparently little staining of the desmosomal plaque proper. Western blots revealed the 08L antigen to be a protein, of M(r) approximately 140,000, found in the Triton-X 100 insoluble pellet. High salt-containing buffers extracted the 08L antigen from the insoluble material. Examination of the assembly of 08L to the desmosome complex, in cells grown in low confluent culture or in calcium-switch assays, by double immunofluorescence with 08L and anti-desmoplakin antibody, revealed that 08L was recruited to morphologically identifiable desmosomes. 08L antigen may exist in a cytosolic pool prior to assembly to the cell surface. The solubility of 08L in low calcium and normal calcium conditions, however, was similar. 08L association to the desmosome was correlated with increased organization of the intermediate filament network. We suggest that the 08L antigen may be involved in the organization and stabilization of the desmosome-IF complexes of epithelia.     

Mechanism of action of the antiplatelet peptide, arietin, from Bitis arietans venom

Arietin, an Arg-Gly-Asp containing peptide from venom of Bitis arietans, inhibited aggregation of platelets stimulated by a variety of agonists with a similar IC50, 1.3-2.7.10(-7) M. It blocked aggregation through the interference of fibrinogen binding to fibrinogen receptors on platelet surface. In this paper, we further demonstrated that arietin had no significant effect on the intracellular mobilization of Ca2+ in Quin2-AM-loaded platelets stimulated by thrombin. It inhibited 125I-fibrinogen binding to ADP-stimulated platelets in a competitive manner (IC50, 1.1.10(-7) M). 125I-arietin bound to unstimulated, ADP-stimulated and elastase-treated platelets in a saturable manner and its Kd values were estimated to be 3.4.10(-7), 3.4.10(-8) and 6.5.10(-8) M, respectively, while the corresponding binding sites were 46,904, 48,958 and 34,817 per platelet, respectively. Arg-Gly-Asp-Ser (RGDS) inhibited 125I-arietin binding to ADP-stimulated platelets in a competitive manner. RGD-containing peptides, including trigramin and rhodostomin, EDTA and monoclonal antibody, 7E3, raised against glycoprotein IIb-IIIa complex, inhibited 125I-arietin binding to ADP-stimulated platelets, indicating that the binding sites of arietin appear to be located at or near glycoprotein IIb-IIIa complex. In conclusion, arietin and other RGD-containing trigramin-like peptides preferentially bind to the fibrinogen receptors associated with glycoprotein IIb-IIIa complex of the activated platelets, thus leading to the blockade of fibrinogen binding to its receptors and subsequent aggregation. The presence of RGD of arietin is essential for the expression of its biological activity. Its binding sites are overlapped with those of trigramin, rhodostomin and the monoclonal antibody, 7E3.     

The role of sphingosine 1‐phosphate and its receptors in cardiovascular diseases

There are many different types of cardiovascular diseases, which impose a huge economic burden due to their extremely high mortality rates, so it is necessary to explore the underlying mechanisms to achieve better supportive and curative care outcomes. Sphingosine 1‐phosphate (S1P) is a bioactive lipid mediator with paracrine and autocrine activities that acts through its cell surface S1P receptors (S1PRs) and intracellular signals. In the circulatory system, S1P is indispensable for both normal and disease conditions; however, there are very different views on its diverse roles, and its specific relevance to cardiovascular pathogenesis remains elusive. Here, we review the synthesis, release and functions of S1P, specifically detail the roles of S1P and S1PRs in some common cardiovascular diseases, and then address several controversial points, finally, we focus on the development of S1P‐based therapeutic approaches in cardiovascular diseases, such as the selective S1PR1 modulator amiselimod (MT‐1303) and the non‐selective S1PR1 and S1PR3 agonist fingolimod, which may provide valuable insights into potential therapeutic strategies for cardiovascular diseases.     

Genetically Encoded FRET Biosensor Detects the Enzymatic Activity of Prostate-Specific Antigen

Prostate cancer is the most common cancer among men beyond 50 years old, and ranked the second in mortality. The level of Prostate-specific antigen (PSA) in serum has been a routine biomarker for clinical assessment of the cancer development, which is detected mostly by antibody-based immunoassays. The proteolytic activity of PSA also has important functions. Here a genetically encoded biosensor based on fluorescence resonance energy transfer (FRET) technology was developed to measure PSA activity. In vitro assay showed that the biosensor containing a substrate peptide ‘RLSSYYSGAG’ had 400% FRET change in response to 1 µg/ml PSA within 90 min, and could detect PSA activity at 25 ng/ml. PSA didn’t show enzymatic activity toward the biosensor in serum solution, likely reflecting the existence of other inhibitory factors besides Zn2+. By expressing the biosensor on cell plasma membrane, the FRET responses were significant, but couldn’t distinguish well the cultured prostate cancer cells from non-prostate cancer cells under microscopy imaging, indicating insufficient speci- ficity to PSA. The biosensor with the previously known ‘HSSKLQ’ substrate showed little response to PSA in solution. In summary, we developed a genetically encoded FRET biosensor to detect PSA activity, which may serve as a useful tool for relevant applications, such as screening PSA activation substrates or inhibitors; the purified biosensor protein can also be an alternative choice for measuring PSA activity besides currently commercialized Mu-HSSKLQ-AMC substrate from chemical synthesis.     

The Effect of Streptococcus salivarius K12 on Halitosis: a Double-Blind,Randomized, Placebo-Controlled Trial

This study was to evaluate the effect of Streptococcus salivarius K12 on tongue coating–associated halitosis. Twenty-eight subjects having tongue coating–associated halitosis were randomly divided into either a test or control group. For each of the 30 days, the test subjects sucked S. salivarius K12 tablet while the control subjects sucked placebo tablets. All the subjects did not take physical (tongue scraping) and chemical (antiseptic mouth-rinse) oral cavity pretreatment prior to use of the tablets. At baseline, and on the 1st, 7th, and 14th day after completing the course of tablets, the subjects were assessed for their organoleptic test (OLT) scores, volatile sulfur compound (VSC) levels, and tongue coating scores (TCS). During the course, all subjects kept their routine oral care habits without scraping their tongue coating. Plaque index, probing depth, and bleeding index were recorded at baseline and at the completion of the trial. On the 1st day following the end of tablet use, the OLT scores and VSC levels had significantly decreased in the test group when compared with the baseline values (P = 0.001 and P = 0.012). The TCS in the test group were also significantly decreased (P = 0.05). At days 7 and 14, the OLT scores in the test group were still significantly lower than the baseline levels (P = 0.006 and P = 0.039 respectively). However, there were no statistical differences with OLT, VSC, and TCS between the test group and the placebo group by analysis of multi-level regression model. The use of S. salivarius K12 did not have significant effect on halitosis with tongue coating cause when the tongue coating was not physically or chemically pre-treated, which implies removing tongue coating is required before Streptococcus salivarius K12 use.

     

Population genetics and sympatric divergence of the freshwater gudgeon,Gobiobotia filifer,in the Yangtze River inferred from mitochondrial DNA

The ecosystem and Pleistocene glaciations play important roles in population demography. The freshwater gudgeon, Gobiobotia filifer, is an endemic benthic fish in the Yangtze River and is a good model for ecological and evolutionary studies. This study aimed to decode the population structure of G. filifer in the Yangtze River and reveal whether divergence occurred before or after population radiation. A total of 292 specimens from eight locations in the upper and middle reaches of the Yangtze River were collected from 2014 to 2016 and analyzed via mitochondrial DNA Cyt b gene sequencing. A moderately high level of genetic diversity was found without structures among the population. However, phylogenetic and network topology showed two distinct haplotype groups, and each group contained a similar proportion of individuals from all sampled sites. This suggested the existence of two genetically divergent source populations in G. filifer. We deduced that a secondary contact of distinct glacial refugia was the main factor creating sympatric populations of G. filifer, and climate improvement promoted population expansion and colonization.     

Gastrodin Alleviates Cerebral Ischaemia/Reperfusion Injury by Inhibiting Pyroptosis by Regulating the lncRNA NEAT1/miR-22-3p Axis

Cerebral ischaemia/reperfusion (I/R) injury-induced irreversible brain injury is a major cause of mortality and functional impairment in ageing people. Gastrodin (GAS), derived from the traditional Chinese herbal medicine Tianma, has been reported to inhibit the progression of stroke, but the mechanism whereby GAS modulates the progression of cerebral I/R remains unclear. The middle cerebral artery occlusion method was used as a model of I/R in vivo. Rats were pretreated with GAS by intraperitoneal injection 7 days before I/R surgery and were then treated with GAS for 7 days after I/R surgery. Additionally, an oxygen–glucose deprivation/reoxygenation model using neuronal cells was established in vitro to simulate I/R injury. 2,3,5-Triphenyltetrazolium chloride and Nissl staining were used to evaluate infarct size and neuronal damage, respectively. Lactate dehydrogenase release and cell counting kit-8 assays were used to assess neuronal cell viability. Enzyme-linked immunosorbent assay, qPCR, flow cytometry and western blotting were performed to analyse the expression levels of inflammatory factors (IL-1β, IL-18), lncRNA NEAT1, miR-22-3p, NLRP3 and cleaved caspase-1. Luciferase reporter experiments were performed to verify the association between lncRNA NEAT1 and miR-22-3p. The results indicated that GAS could significantly improve the neurological scores of rats and reduce the area of cerebral infarction. Meanwhile, GAS inhibited pyroptosis by downregulating NLRP3, inflammatory factors (IL-1β, IL-18) and cleaved caspase-1. In addition, GAS attenuated I/R-induced inflammation in neuronal cells through the modulation of the lncRNA NEAT1/miR-22-3p axis. GAS significantly attenuated cerebral I/R injury via modulation of the lncRNA NEAT1/miR-22-3p axis. Thus, GAS might serve as a new agent for the treatment of cerebral I/R injury.