An Extracellullar Nuclease from Bacillus Firmus VKPACU-1: Specificity and Mode of Action

An extracellular nuclease from Bacillus firmus VKPACU-1 was multifunctional enzyme, this nuclease hydrolyzed poly U rapidly and more preferentially than the other homopolyribonucleotides. Hydrolysis of RNA this enzyme released mononucleotides in the order 5′UMP > 5′AMP > 5′GMP where as in hydrolysis of DNA the mononucleotides in the order of 5′dAMP > 5′dGMP > 5′dTMP and oligonucleotides. Uridylic linkages in RNA and adenylic linkages in DNA were preferentially cleaved by the nuclease. Nuclease produced oligonucleotides having only 3’ hydroxyl and 5’ phosphate termini. Present nuclease hydrolyzed RNA and DNA released oligonucleotides as major end products and mononucleotides, suggesting an endo mode of action.     

Estrogen Receptor β Signaling Induces Autophagy and Downregulates Glut9 Expression

Glut9 is highly expressed in the human kidney proximal convoluted tubular and plays a crucial role in the regulation of plasma urate levels. The gene effects were stronger among women. Our results show that 17-β-estradiol (E2) through ER (estrogen receptor) β downregulates Glut9 protein expression on human renal tubular epithelial cell line (HK2). Intriguingly, E2 does not affect the expression of Glut9 mRNA. ERβ is linked to PTEN, the PTEN gene negatively regulates the PI3K/AKT pathway, and the PI3K/AKT pathway inhibition may lead to autophagy. Further study indicates that ERβ may affect the expression of Glut9 though autophagy.     

Characterization of bacteriocin bificin C6165: a novel bacteriocin

Aims

To purify and primarily characterize an anti‐Alicyclobacillus bacteriocin produced by Bifidobacterium animalis subsp. animalis CICC 6165, suggested to be named bificin C6165.

Methods and Results

During purification of the bificin C6165, optimal recovery was achieved with ammonium sulfate precipitation followed by two chromatographic steps. Mass spectrometry analyses revealed a distinctive peak corresponding to a molecular mass of 3395·1 Da. This bacteriocin was heat stable, effective after refrigerated storage and freeze–thaw cycles. The primary mode of action of bificin C6165 is most probably due to pore formation, as indicated by the efflux of K⁺ from metabolically active cells of Alicyclobacillus acidoterrestris. In the presence of 10 mmol l^?1 gadolinium, bificin C6165 did not affect cells of Alicyclobacillus acidoterrestris. This suggests that the mode of action of bificin C6165 relies on a net negatively charged cell surface.

Conclusions

Bificin C6165 is indeed a novel bacteriocin and it exhibited remarkable potency for Alicyclobacillus control.

Significance and Impact of the Study

Application of bacteriocins in preservation of fruit juices has seldom been studied. Bificin C6165 may be an alternative method to control juice spoilage by this Alicyclobacillus acidoterrestris and meet increasing consumer demand for nature and artificial chemical additive‐free food products.     

Subtype Identification in Acutely Dissociated Rat Nodose Ganglion Neurons Based on Morphologic Parameters

Nodose ganglia are composed of A-, Ah- and C-type neurons. Despite their important roles in regulating visceral afferent function, including cardiovascular, pulmonary, and gastrointestinal homeostasis, information about subtype-specific expression, molecular identity, and function of individual ion transporting proteins is scarce. Although experiments utilizing the sliced ganglion preparation have provided valuable insights into the electrophysiological properties of nodose ganglion neuron subtypes, detailed characterization of their electrical phenotypes will require measurements in isolated cells. One major unresolved problem, however, is the difficulty to unambiguously identify the subtype of isolated nodose ganglion neurons without current-clamp recording, because the magnitude of conduction velocity in the corresponding afferent fiber, a reliable marker to discriminate subtypes in situ, can no longer be determined. Here, we present data supporting the notion that application of an algorithm regarding to microscopic structural characteristics, such as neuron shape evaluated by the ratio between shortest and longest axis, neuron surface characteristics, like membrane roughness, and axon attachment, enables specific and sensitive subtype identification of acutely dissociated rat nodose ganglion neurons, by which the accuracy of identification is further validated by electrophysiological markers and overall positive predictive rates is 89.26% (90.04%, 76.47%, and 98.21% for A-, Ah, and C-type, respectively). This approach should aid in gaining insight into the molecular correlates underlying phenotypic heterogeneity of nodose ganglia. Additionally, several critical points that help for neuron identification and afferent conduction calibration are also discussed.     

Upregulation of KCNQ1/KCNE1 K+ channels by Klotho

Klotho is a transmembrane protein expressed primarily in kidney, parathyroid gland, and choroid plexus. The extracellular domain could be cleaved off and released into the systemic circulation. Klotho is in part effective as β-glucuronidase regulating protein stability in the cell membrane. Klotho is a major determinant of aging and life span. Overexpression of Klotho increases and Klotho deficiency decreases life span. Klotho deficiency may further result in hearing loss and cardiac arrhythmia. The present study explored whether Klotho modifies activity and protein abundance of KCNQ1/KCNE1, a K⁺ channel required for proper hearing and cardiac repolarization. To this end, cRNA encoding KCNQ1/KCNE1 was injected in Xenopus oocytes with or without additional injection of cRNA encoding Klotho. KCNQ1/KCNE1 expressing oocytes were treated with human recombinant Klotho protein (30 ng/ml) for 24 h. Moreover, oocytes which express both KCNQ1/KCNE1 and Klotho were treated with 10 µM DSAL (D-saccharic acid-1,4-lactone), a β-glucuronidase inhibitor. The KCNQ1/KCNE1 depolarization-induced current (I_Ks) was determined utilizing dual electrode voltage clamp, while KCNQ1/KCNE1 protein abundance in the cell membrane was visualized utilizing specific antibody binding and quantified by chemiluminescence. KCNQ1/KCNE1 channel activity and KCNQ1/KCNE1 protein abundance were upregulated by coexpression of Klotho. The effect was mimicked by treatment with human recombinant Klotho protein (30 ng/ml) and inhibited by DSAL (10 µM). In conclusion, Klotho upregulates KCNQ1/KCNE1 channel activity by 'mainly' enhancing channel protein abundance in the plasma cell membrane, an effect at least partially mediated through the β-glucuronidase activity of Klotho protein.     

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.     

Beautiful,complicated—and intelligent? Novel aspects of the thigmonastic stamen movement in Loasaceae

In a recent study we investigated the complex mechanisms regulating the pollen release via thigmonastic stamen movement found exclusively in Loasaceae subfamily Loasoideae. We demonstrated that stamen movement is modulated by abiotic (light and temperature) as well as biotic stimuli (pollinator availability and visitation frequency). This is explained as a mechanism to adjust the rate of stamen movement and thus pollen dispensation to different environmental conditions in order to optimize pollen transfer. Stamen movement is rapid and thus a near-immediate response to pollinator visits. However, Loasaceae flowers also show a response to biotic stimuli on a longer time scale, by adjusting the duration of both the staminate and the carpellate phase of the anthesis. We here present two additional data sets on species not previously studied, underscoring the shortening of the staminate phase in the presence of pollinator visits vs. their absence and the shortening of the carpellate phase after pollination. Overall, the plant shows not only a rapid but an “intelligent” reaction to its environment in adjusting anthesis and pollen presentation to a range of factors. The physiological and morphological bases of the stamen movement are poorly understood. Our previous study showed that there is no direct spatial relationship between the place of stimulation in the flower and the stamen bundle activated. We here further show the morphological basis for stamen movement from a reflexed into an erect position: Only the basal part of the filament curves around the receptacle, while the upper part of the filament retains its shape. We hypothesize that the stimulus is transmitted over the entire receptacle and the place of reaction is determined by stamen maturity, not the location of the stimulus.     

Unexpected mode of action of sweet potato β-amylase on maltooligomer substrates

β-Amylase (EC 3.2.1.2), one of the main protein of the sweet potato, is an exo-working enzyme catalyzing the hydrolysis of α(1,4) glycosidic linkages in polysaccharides and removes successively maltose units from the non-reducing ends. The enzyme belongs to glycoside hydrolase GH14 family and inverts the anomeric configuration of the hydrolysis product. Multiple attack or processivity is an important property of polymer active enzymes and there is still limited information about the processivity of carbohydrate active enzymes. Action pattern and kinetic measurements of sweet potato β-amylase were made on a series of aromatic chromophor group-containing substrates (degree of polymerization DP 3-13) using HPLC method. Measured catalytic efficiencies increased with increasing DP of the substrates. Processive cleavage was observed on all substrates except the shortest pentamer. The mean number of steps without dissociation of enzyme–product complex increases with DP of substrate and reached 3.3 in case of CNPG11 indicating that processivity on longer substrates was more significant. A unique transglycosylation was observed on those substrates, which suffer processive cleavage and the substrates were re-built by the enzyme. Our results are the first presentation of a transglycosylation during an inverting glycosidase catalyzed hydrolysis. The yield of transglycosylation was remarkable high as shown in the change of the CNPG11 quantity. The CNPG11 concentration was doubled (from 0.24 to 0.54 mM) in the early phase of the reaction.     

Bindings of PPARγ ligand-binding domain with 5-cholesten-3β, 25-diol, 3-sulfate: accurate prediction by molecular simulation

Abstract

Peroxisome proliferator-activated receptor gamma (PPAR_γ) has recently been identified as an attractive target for atherosclerosis intervention. Given potential relevance of 5-cholesten-3β, 25-diol, 3-sulphate (CHOS) and PPAR_γ, an integrated docking method was used to study their interaction mechanisms, with the full considerations to distinct CHOS conformations and dynamic ensembles of PPAR_γ ligand-binding domain (PPAR_γ-LBD). The results revealed that this novel platform is satisfactory to the accurate determination of binding profiles, and the binding pattern of CHOS is rather similar as those of current PPAR_γ full/partial agonists. CHOS contributes to the stabilization of the AF2 and β-sheet surfaces of PPAR_γ-LBD and promotes the configuration adjustment of Ω loop, in order to inhibit the Cdk5-mediated PPAR_γ phosphorylation. Nonetheless, there are clear differences in term of occupation of full or partial agonist-like binding models. The energetic and geometric analyses further revealed that CHOS may be fond of partial agonist-like binding, and its sulfonic group and carbon skeleton are helpful for the binding process. We hope that the results will aid our understanding of recognitions involving CHOS with PPAR_γ-LBD and warrant the further aspects to pharmacological experiments.

Communicated by Ramaswamy H. Sarma