Transglucosidic reactions of the Aspergillus niger family 3 beta-glucosidase: qualitative and quantitative analyses and evidence that the transglucosidic rate is independent of pH

The hydrolytic and transglucosidic reactions of the Aspergillus niger Family 3 beta-glucosidase were characterized. Michaelis-Menten plots of the rates of aglycone formation were normal (hyperbolic) at low [substrate]. However, at high [substrate] the rates decreased at pH below approximately 5.5 but increased at pH above approximately 5.5. Each decrease or increase took the form of a second hyperbola adjoining the first. Thin layer chromatography, gas-liquid chromatography, and NMR analyses indicated that the substrates became transglucosidic acceptors when present at high concentrations. When pNPGlc and cellobiose reacted as acceptors, the C6 hydroxyl of the non-reducing substrate component reacted to form beta-D-glucopyranosyl-(1-6)-beta-D-glucopyranosyl-p-nitrophenol and beta-D-glucopyranosyl-(1-6)-beta-D-glucopyranosyl-(1-4)-D-glucopyranose, respectively. The acceptor action accounted for the second adjoining hyperbolas. Rate equations were derived for the production of the aglycone and the transglucosidic intermediate, and these equations described the data very well. Hydrolytic Vmax {Vmax(h)}, hydrolytic Km {Km(h)}, transglucosidic Vmax {Vmax(t)}, and transglucosidic Km {Km(t)} values were obtained by non-linear regression analysis using these equations. Vmax(h) pH profiles were bell shaped with optima between pH 4 and 4.5 but the Vmax(t) values did not change substantially between pH 3 and 7. These differences in the pH profiles explain the decreasing and increasing adjoining hyperbolas since Vmax(t) is lower than Vmax(h) at pH less than approximately 5.5 but higher than Vmax(h) at pH greater than approximately 5.5. The reason for these pH effects is that the value of the hydrolytic rate constant (k3) decreases while the value of the transglucosidic rate constant (k4) does not change between pH 3 and 7. The study also showed that gentiobiose forms by an intermolecular reaction of the C6 hydroxyl of Glc rather than an intramolecular reaction and that an equatorial orientation of the C2 hydroxyl, the presence of a C6 primary hydroxyl and beta-linkages with oligosaccharide acceptors are important for acceptor reactivity.     

Synergistic interaction between Gdf1 and Nodal during anterior axis development

Growth and Differentiation Factor 1 (GDF-1) has been implicated in left-right patterning of the mouse embryo but has no other known function. Here, we demonstrate a genetic interaction between Gdf1 and Nodal during anterior axis development. Gdf1-/-;Nodal+/- mutants displayed several abnormalities that were not present in either Gdf1-/- or Nodal+/- single mutants, including absence of notochord and prechordal plate, and malformation of the foregut; organizing centers implicated in the development of the anterior head and branchial arches, respectively. Consistent with these deficits, Gdf1-/-;Nodal+/- mutant embryos displayed a number of axial midline abnormalities, including holoprosencephaly, anterior head truncation, cleft lip, fused nasal cavity, and lack of jaws and tongue. The absence of these defects in single mutants indicated a synergistic interaction between Nodal and GDF-1 in the node, from which the axial mesendoderm that gives rise to the notochord, prechordal plate, and foregut endoderm originates, and where the two factors are co-expressed. This notion was supported by a severe downregulation of FoxA2 and goosecoid in the anterior primitive streak of double mutant embryos. Unlike that in the lateral plate mesoderm, Nodal expression in the node was independent of GDF-1, indicating that both factors act in parallel to control the development of mesendodermal precursors. Receptor reconstitution experiments indicated that GDF-1, like Nodal, can signal through the type I receptors ALK4 and ALK7. However, analysis of compound mutants indicated that ALK4, but not ALK7, was responsible for the effects of GDF-1 and Nodal during anterior axis development. These results indicate that GDF-1 and Nodal converge on ALK4 in the anterior primitive streak to control the formation of organizing centers that are necessary for normal forebrain and branchial arch development.     

Adenosine analogue–oligo-arginine conjugates (ARCs) serve as high-affinity inhibitors and fluorescence probes of type I cGMP-dependent protein kinase (PKGIα)

Introduction

Type I cGMP-dependent protein kinase (PKGIα) belongs to the family of cyclic nucleotide-dependent protein kinases and is one of the main effectors of cGMP. PKGIα is involved in regulation of cardiac contractility, vasorelaxation, and blood pressure; hence, the development of potent modulators of PKGIα would lead to advances in the treatment of a variety of cardiovascular diseases. Aim: Representatives of ARC-type compounds previously characterized as potent inhibitors and high-affinity fluorescent probes of PKA catalytic subunit (PKAc) were tested towards PKGIα to determine that ARCs could serve as activity regulators and sensors for the latter protein kinase both in vitro and in complex biological systems. Results: Structure–activity profiling of ARCs with PKGIα in vitro demonstrated both similarities as well as differences to corresponding profiling with PKAc, whereas ARC-903 and ARC-668 revealed low nanomolar displacement constants and inhibition IC₅₀ values with both cyclic nucleotide-dependent kinases. The ability of ARC-based fluorescent probes to penetrate cell plasma membrane was demonstrated in the smooth muscle tissue of rat cerebellum isolated arteries, and the compound with the highest affinity in vitro (ARC-903) showed also potential for in vivo applications, fully abolishing the PKG1α-induced vasodilation.     

Effects of β1 →4 Linked Galactooligosaccharides on Use of Magnesium and Calcification of the Kidney and Heart in Rats Fed Excess Dietary Phosphorus and Calcium

Magnesium deficiency was induced in male Wistar rats by adding an excess of phosphorus and calcium to the diet (1.195 g of phosphorus and 1.04g of calcium/100g of diet). Feeding of these animals with a diet containing β1→4 linked galactooligosaccharides (4′-GOS) (5g of 4′-GOS/100g of diet) increased the apparent magnesium absorption ratios and the concentrations of magnesium in the serum and femur, and reduced accumulation of calcium in the kidney and heart. We speculate that the use of magnesium increased by feeding 4′-GOS to a limited extent prevented the lower magnesium status and the severity of calcification of the kidney and heart caused by excess dietary phosphorus and calcium.     

Design,synthesis and biological evaluation of novel anti-cytokine 1,2,4-triazine derivatives

A series of 16 novel 1,2,4-triazine derivatives bearing hydrazone moiety (7a–7p) have been designed, synthesized and evaluated for their activity to inhibit IL-1β and TNF-α production. All compounds are reported for the first time. The chemical structures of all compounds were confirmed by spectroscopic methods and elemental analyzes. Most of the synthesized compounds were proved to have potent anti-cytokine activity and low toxicity on PBMC and MCF-7 cell lines. Compounds 7f, 7k, 7l and 7j presented simultaneously good levels of inhibition of both cytokines. Moreover, compound 7l exhibited good anti-inflammatory effect in carrageenan-induced rat paw edema. The results of Western blotting demonstrated that the anti-cytokine potential of compound 7l is mainly mediated through the inhibition of p38 MAPK signaling pathway. Molecular docking was performed to position compound 7l into p38α binding site in order to explore the potential target. The information of this work might be helpful for the design and synthesis of novel scaffold toward the development of new therapeutic agent to fight against inflammatory diseases.     

DNA repair mechanisms in dividing and non-dividing cells

DNA damage created by endogenous or exogenous genotoxic agents can exist in multiple forms, and if allowed to persist, can promote genome instability and directly lead to various human diseases, particularly cancer, neurological abnormalities, immunodeficiency and premature aging. To avoid such deleterious outcomes, cells have evolved an array of DNA repair pathways, which carry out what is typically a multiple-step process to resolve specific DNA lesions and maintain genome integrity. To fully appreciate the biological contributions of the different DNA repair systems, one must keep in mind the cellular context within which they operate. For example, the human body is composed of non-dividing and dividing cell types, including, in the brain, neurons and glial cells. We describe herein the molecular mechanisms of the different DNA repair pathways, and review their roles in non-dividing and dividing cells, with an eye toward how these pathways may regulate the development of neurological disease.     

Alleviating the inhibitory effect of salinity stress on nod gene expression in Rhizobium tibeticum – fenugreek (Trigonella foenum graecum) symbiosis by isoflavonoids treatment

Rhizobia-legume symbiosis depends on molecular dialog, which involves the production of specific plant flavonoid compounds as signal molecules. Rhizobium tibeticum was recovered from the root nodule of fenugreek and identified by sequencing the 16S rRNA gene. The effect of salinity stress on nod gene expression was measured in terms of β-galactosidase activity. R. tibeticum containing Escherichia coli lacZ gene fusions to specific nodulation (nod) genes were used to determine β-galactosidase activity. Combination of hesperetin (7.5 µM) and apigenin (7.5 µM) significantly increased β-galactosidase activity more than the single application of hesperetin or apigenin. Preincubation of R. tibeticum with hesperetin and apigenin combination significantly alleviates the adverse effect of salinity on nod gene expression and therefore, enhances nodulation and nitrogen fixation of fenugreek.     

Brain mineralocorticoid receptors in cognition and cardiovascular homeostasis

Mineralocorticoid receptors (MR) mediate diverse functions supporting osmotic and hemodynamic homeostasis, response to injury and inflammation, and neuronal changes required for learning and memory. Inappropriate MR activation in kidneys, heart, vessels, and brain hemodynamic control centers results in cardiovascular and renal pathology and hypertension. MR binds aldosterone, cortisol and corticosterone with similar affinity, while the glucocorticoid receptor (GR) has less affinity for cortisol and corticosterone. As glucocorticoids are more abundant than aldosterone, aldosterone activates MR in cells co-expressing enzymes with 11β-hydroxydehydrogenase activity to inactivate them. MR and GR co-expressed in the same cell interact at the molecular and functional level and these functions may be complementary or opposing depending on the cell type. Thus the balance between MR and GR expression and activation is crucial for normal function. Where 11β-hydroxydehydrogenase 2 (11β-HSD2) that inactivates cortisol and corticosterone in aldosterone target cells of the kidney and nucleus tractus solitarius (NTS) is not expressed, as in most neurons, MR are activated at basal glucocorticoid concentrations, GR at stress concentrations. An exception may be pre-autonomic neurons of the PVN which express MR and 11β-HSD1 in the absence of hexose-6-phosphate dehydrogenase required to generate the requisite cofactor for reductase activity, thus it acts as a dehydrogenase. MR antagonists, valuable adjuncts to the treatment of cardiovascular disease, also inhibit MR in the brain that are crucial for memory formation and exacerbate detrimental effects of excessive GR activation on cognition and mood. 11β-HSD1 inhibitors combat metabolic and cognitive diseases related to glucocorticoid excess, but may exacerbate MR action where 11β-HSD1 acts as a dehydrogenase, while non-selective 11β-HSD1&2 inhibitors cause injurious disruption of MR hemodynamic control. MR functions in the brain are multifaceted and optimal MR:GR activity is crucial. Therefore selectively targeting down-stream effectors of MR specific actions may be a better therapeutic goal.     

Conversion of 4-androstene-3,17-dione to testosterone by Pisum sativum

4-Androstene-3,17-dione-[4-¹⁴C] was applied to the leaves of growing pea plants, Pisum sativum. Within a week, 28% of the administered steroid was specifically reduced to testosterone. Part of the testosterone was present in esterified form, and 5α-androstane-3β,17β-diol was also identified as a metabolite, but neither epitestosterone nor estrogens were detected.     

Methyl-β-cyclodextrin potentiates the BITC-induced anti-cancer effect through modulation of the Akt phosphorylation in human colorectal cancer cells

ABSTRACT

Methyl-β-cyclodextrin (MβCD) is an effective agent for the removal of plasma membrane cholesterol. In this study, we investigated the modulating effects of MβCD on the antiproliferation induced by benzyl isothiocyanate (BITC), an ITC compound mainly derived from papaya seeds. We confirmed that MβCD dose-dependently increased the cholesterol level in the medium, possibly through its removal from the plasma membrane of human colorectal cancer cells. The pretreatment with a non-toxic concentration (2.5 mM) of MβCD significantly enhanced the BITC-induced cytotoxicity and apoptosis induction, which was counteracted by the cholesterol supplementation. Although BITC activated the phosphoinositide 3-kinase (PI3K)/Akt pathway, MβCD dose-dependently inhibited the phosphorylation level of Akt. On the contrary, the treatment of MβCD enhanced the phosphorylation of mitogen activated protein kinases, but did not potentiate their BITC-induced phosphorylation. These results suggested that MβCD might potentiate the BITC-induced anti-cancer by cholesterol depletion and thus inhibition of the PI3K/Akt-dependent survival pathway.

Abbreviations: CDs: cyclodextrins; MβCD: methyl-β-cyclodextrin; ITCs: isothiocyanates; BITC: benzyl isothiocyanate; PI3K: phosphoinositide 3-kinase; PDK1: phosphoinositide-dependent kinase-1; MAPK: mitogen activated protein kinase; ERK1/2: extracellular signal-regulated kinase1/2; JNK: c-Jun N-terminal kinase; PI: propidium iodide; FBS: fatal bovine serum; TLC: thin-layer chromatography; PBS(-): phosphate-buffered saline without calcium and magnesium; MEK: MAPK/ERK kinase; PIP2: phosphatidylinositol-4,5-bisphosphate; PIP3: phosphatidylinositol-3,4,5-trisphosphate