Anti-Hyperglycemic Effects of Oils and Extracts Derived from Sea Buckthorn - A Comprehensive Analysis Utilizing In Vitro and In Vivo Models.

SCOPE: Sea buckthorn (Hippophaes rhamnoides) is capable of ameliorating disturbed glucose metabolism in animal models and human subjects. Here, the effect of sea buckthorn oil as well as of extracts of fruits, leaves, and press cake on postprandial glucose metabolism is systematically investigated. METHODS AND RESULTS: Sea buckthorn did neither exert decisive effects in an in vitro model of intestinal glucose absorption nor did it alter insulin secretion. However, sea buckthorn stimulates GLUT4 translocation to the plasma membrane comparable to insulin, indicative of increased glucose clearance from the circulation. Isorhamnetin is identified in all sea buckthorn samples investigated and is biologically active in triggering GLUT4 cell surface localization. Consistently, sea buckthorn products lower circulating glucose by ≈10% in a chick embryo model. Moreover, sea buckthorn products fully revert hyperglycemia in the nematode Caenorhabditis elegans while they are ineffective in Drosophila melanogaster under euglycemic conditions. CONCLUSION: These data indicate that edible sea buckthorn products as well as by-products are promising resources for hypoglycemic nutrient supplements that increase cellular glucose clearance into target tissues.

Metabolite and Bioactive Compounds Profiling of Meteora Sea Buckthorn Berries through High-Resolution NMR Analysis.

Sea buckthorn berries (Hippophaë rhamnoides L.) (SB) are considered as a fruit with a high nutritional value with a plethora of bioactive ingredients. The present work focusses on the analysis of the whole NMR metabolic profile of SB berries grown in an organic orchard of Meteora/Greece. In parallel, this study validates/highlights qualitative characteristics of the osmotic processed berries according to the fresh fruit. The composition in bioactive metabolites of SB berries was elucidated through sophisticated high-resolution NMR spectroscopy. The lipophilic profile maintains the vitamins, flavonoid glycosides, phenolic esters and the essential lipid components of SB, while the polar profile reveals a variety of flavonoids, saccharides, organic acids, amino acids and esterified glycosides. This approach towards identification of SB bioactive ingredients may serve as basis for simultaneous profiling and quality assessment and may be applied to monitor fresh food quality regarding other food preservation methods.

A quorum sensing small volatile molecule promotes antibiotic tolerance in bacteria.

Bacteria can be refractory to antibiotics due to a sub-population of dormant cells, called persisters that are highly tolerant to antibiotic exposure. The low frequency and transience of the antibiotic tolerant "persister" trait has complicated elucidation of the mechanism that controls antibiotic tolerance. In this study, we show that 2' Amino-acetophenone (2-AA), a poorly studied but diagnostically important small, volatile molecule produced by the recalcitrant gram-negative human pathogen Pseudomonas aeruginosa, promotes antibiotic tolerance in response to quorum-sensing (QS) signaling. Our results show that 2-AA mediated persister cell accumulation occurs via alteration of the expression of genes involved in the translational capacity of the cell, including almost all ribosomal protein genes and other translation-related factors. That 2-AA promotes persisters formation also in other emerging multi-drug resistant pathogens, including the non 2-AA producer Acinetobacter baumannii implies that 2-AA may play an important role in the ability of gram-negative bacteria to tolerate antibiotic treatments in polymicrobial infections. Given that the synthesis, excretion and uptake of QS small molecules is a common hallmark of prokaryotes, together with the fact that the translational machinery is highly conserved, we posit that modulation of the translational capacity of the cell via QS molecules, may be a general, widely distributed mechanism that promotes antibiotic tolerance among prokaryotes.

Hydrogen sulfide-mediated stimulation of mitochondrial electron transport involves inhibition of the mitochondrial phosphodiesterase 2A, elevation of cAMP and activation of protein kinase A.

Although hydrogen sulfide (H2S) is generally known as a mitochondrial poison, recent studies show that lower concentrations of H2S play a physiological role in the stimulation of mitochondrial electron transport and cellular bioenergetics. This effect involves electron donation at Complex II. Other lines of recent studies demonstrated that one of the biological actions of H2S involves inhibition of cAMP and cGMP phosphodiesterases (PDEs). Given the emerging functional role of the mitochondrial isoform of cAMP PDE (PDE2A) in the regulation of mitochondrial function the current study investigated whether cAMP-dependent mechanisms participate in the stimulatory effect of NaHS on mitochondrial function. In isolated rat liver mitochondria, partial digestion studies localized PDE2A into the mitochondrial matrix. NaHS exerted a concentration-dependent inhibitory effect on recombinant PDE2A enzyme in vitro. Moreover, NaHS induced an elevation of cAMP levels when added to isolated mitochondria and stimulated the mitochondrial electron transport. The latter effect was inhibited by Rp-cAMP, an inhibitor of the cAMP-dependent protein kinase (PKA). The current findings suggest that the direct electron donating effect of NaHS is amplified by an intramitochondrial cAMP system, which may involve the inhibition of PDE2A and subsequent, cAMP-mediated stimulation of PKA.

Selectivity of commonly used pharmacological inhibitors for cystathionine ß synthase (CBS) and cystathionine γ lyase (CSE).

BACKGROUND AND PURPOSE: Hydrogen sulfide (H2S) is a signalling molecule that belongs to the gasotransmitter family. Two major sources for endogenous enzymatic production of H2S are cystathionine ß synthase (CBS) and cystathionine γ lyase (CSE). In the present study, we examined the selectivity of commonly used pharmacological inhibitors of H2S biosynthesis towards CSE and CBS. EXPERIMENTAL APPROACH: To address this question, human CSE or CBS enzymes were expressed and purified from Escherichia coli as fusion proteins with GSH-S-transferase. After purification, the activity of the recombinant enzymes was tested using the methylene blue method. KEY RESULTS: ß-Cyanoalanine (BCA) was more potent in inhibiting CSE than propargylglycine (PAG) (IC50 14 ± 0.2 µM vs. 40 ± 8 µM respectively). Similar to PAG, L-aminoethoxyvinylglycine (AVG) only inhibited CSE, but did so at much lower concentrations. On the other hand, aminooxyacetic acid (AOAA), a frequently used CBS inhibitor, was more potent in inhibiting CSE compared with BCA and PAG (IC50 1.1 ± 0.1 µM); the IC50 for AOAA for inhibiting CBS was 8.5 ± 0.7 µM. In line with our biochemical observations, relaxation to L-cysteine was blocked by AOAA in aortic rings that lacked CBS expression. Trifluoroalanine and hydroxylamine, two compounds that have also been used to block H2S biosynthesis, blocked the activity of CBS and CSE. Trifluoroalanine had a fourfold lower IC50 for CBS versus CSE, while hydroxylamine was 60-fold more selective against CSE. CONCLUSIONS AND IMPLICATIONS: In conclusion, although PAG, AVG and BCA exhibit selectivity in inhibiting CSE versus CBS, no selective pharmacological CBS inhibitor is currently available.

Hydrogen sulfide and nitric oxide are mutually dependent in the regulation of angiogenesis and endothelium-dependent vasorelaxation.

Hydrogen sulfide (H(2)S) is a unique gasotransmitter, with regulatory roles in the cardiovascular, nervous, and immune systems. Some of the vascular actions of H(2)S (stimulation of angiogenesis, relaxation of vascular smooth muscle) resemble those of nitric oxide (NO). Although it was generally assumed that H(2)S and NO exert their effects via separate pathways, the results of the current study show that H(2)S and NO are mutually required to elicit angiogenesis and vasodilatation. Exposure of endothelial cells to H(2)S increases intracellular cyclic guanosine 5'-monophosphate (cGMP) in a NO-dependent manner, and activated protein kinase G (PKG) and its downstream effector, the vasodilator-stimulated phosphoprotein (VASP). Inhibition of endothelial isoform of NO synthase (eNOS) or PKG-I abolishes the H(2)S-stimulated angiogenic response, and attenuated H(2)S-stimulated vasorelaxation, demonstrating the requirement of NO in vascular H(2)S signaling. Conversely, silencing of the H(2)S-producing enzyme cystathionine-γ-lyase abolishes NO-stimulated cGMP accumulation and angiogenesis and attenuates the acetylcholine-induced vasorelaxation, indicating a partial requirement of H(2)S in the vascular activity of NO. The actions of H(2)S and NO converge at cGMP; though H(2)S does not directly activate soluble guanylyl cyclase, it maintains a tonic inhibitory effect on PDE5, thereby delaying the degradation of cGMP. H(2)S also activates PI3K/Akt, and increases eNOS phosphorylation at its activating site S1177. The cooperative action of the two gasotransmitters on increasing and maintaining intracellular cGMP is essential for PKG activation and angiogenesis and vasorelaxation. H(2)S-induced wound healing and microvessel growth in matrigel plugs is suppressed by pharmacological inhibition or genetic ablation of eNOS. Thus, NO and H(2)S are mutually required for the physiological control of vascular function.

Antisense masking reveals contributions of mRNA-rRNA base pairing to translation of Gtx and FGF2 mRNAs.

We previously showed that a 9-nucleotide sequence from the 5' leader of the Gtx homeodomain mRNA facilitates translation initiation by base pairing to 18S rRNA. These earlier studies tested the Gtx element in isolation; we now assess the physiological relevance of this element in the context of two natural mRNAs that contain this sequence in their 5' leaders, Gtx itself and FGF2 (fibroblast growth factor 2). 2'-O-Methyl-modified RNA oligonucleotides were employed to block mRNA-rRNA base pairing by targeting either the Gtx-binding site in 18S rRNA or Gtx elements in recombinant mRNAs containing the Gtx or FGF2 5' leaders linked to a reporter cistron. Studies in cell-free lysates and transfected COS-7 cells showed that translation of mRNAs containing the Gtx or FGF2 5' leaders was decreased by > 50% when oligonucleotides targeting either the rRNA or mRNA were used. Specificity was demonstrated by showing that translation of the recombinant mRNAs was unaffected by control oligonucleotides. In addition, the specific oligonucleotides did not affect the translation of recombinant mRNAs in which the Gtx elements were mutated. Experiments performed using constructs containing Gtx and FGF2 5' leader and coding sequences ruled out possible effects of the reporter cistron. Furthermore, two-dimensional gel electrophoresis revealed that the oligonucleotides used in this study had little overall effect on the proteomes of cells transfected with these oligonucleotides. This study demonstrates that mRNA-rRNA base pairing affects the expression of two cellular mRNAs and describes a new approach for investigating putative mRNA-rRNA base pairing interactions in mammalian cells.

Eukaryotic ribosomal proteins lacking a eubacterial counterpart: important players in ribosomal function.

The ribosome is a macromolecular machine responsible for protein synthesis in all organisms. Despite the enormous progress in studies on the structure and function of prokaryotic ribosomes, the respective molecular details of the mechanism by which the eukaryotic ribosome and associated factors construct a polypeptide accurately and rapidly still remain largely unexplored. Eukaryotic ribosomes possess more RNA and a higher number of proteins than eubacterial ribosomes. As the tertiary structure and basic function of the ribosomes are conserved, what is the contribution of these additional elements? Elucidation of the role of these components should provide clues to the mechanisms of translation in eukaryotes and help unravel the molecular mechanisms underlying the differences between eukaryotic and eubacterial ribosomes. This article focuses on a class of eukaryotic ribosomal proteins that do not have a eubacterial homologue. These proteins play substantial roles in ribosomal structure and function, and in mRNA binding and nascent peptide folding. The role of these proteins in human diseases and viral expression, as well as their potential use as targets for antiviral agents is discussed.

Biochemical evidence of translational infidelity and decreased peptidyltransferase activity by a sarcin/ricin domain mutation of yeast 25S rRNA.

A C-->U mutation (rdn5) in the conserved sarcin/ricin domain of yeast 25S rRNA has been shown to cause translational suppression and paromomycin resistance. It also separates the killing from the misreading effect of this antibiotic. We confirm these findings and provide in vitro evidence that rdn5 causes a 3-fold increase in translational errors and resistance to paromomycin. The role of this 25S rRNA domain in ribosome's decoding function was further demonstrated when 60S subunits from rdn5 cells were combined with 40S subunits from cells carrying an error-prone mutation in the eukaryotic accuracy center ribosomal protein S23, an homologue of Escherichia coli S12. These hybrids exhibited an error frequency similar to that of rdn5 alone, despite the error-prone mutation in S23. This was accompanied by extreme resistance to paromomycin, unlike the effects of the individual mutations. Furthermore, rdn5 lowers peptidyltransferase activity measured as a second-order rate constant (kcat/K(s)) corresponding to the rate of peptide bond formation. This mutation was also found to affect translocation. Elongation factor 2 (EF2)-dependent translocation of Ac-Phe-tRNA from the A- to P-site was achieved at an EF2 concentration 3.5 times lower than in wild type. In conclusion, the sarcin/ricin domain of 25S rRNA influences decoding, peptide bond formation and translocation.

A dispensable yeast ribosomal protein optimizes peptidyltransferase activity and affects translocation.

Yeast ribosomal protein L41 is dispensable in the yeast. Its absence had no effect on polyphenylalanine synthesis activity, and a limited effect on growth, translational accuracy, or the resistance toward the antibiotic paromomycin. Removal of L41 did not affect the 60:40 S ratio, but it reduced the amount of 80 S, suggesting that L41 is involved in ribosomal subunit association. However, the two most important effects of L41 were on peptidyltransferase activity and translocation. Peptidyltransferase activity was measured as a second-order rate constant (k(cat)/K(s)) corresponding to the rate of peptide bond formation; this k(cat)/K(s) was lowered 3-fold to 1.15 min(-1) mm(-1) in the L41 mutant compared with 3.46 min(-1) mm(-1) in the wild type. Translocation was also affected by L41. Elongation factor 2 (EF2)-dependent (enzymatic) translocation of Ac-Phe-tRNA from the A- to P-site was more efficient in the absence of L41, because 50% translocation was achieved at only 0.004 microm EF2 compared with 0.02 microm for the wild type. Furthermore, the EF2-dependent translocation was inhibited by 50% at 2.5 microm of the translocation inhibitor cycloheximide in the L41 mutant compared with 1.2 microm in the wild type. Finally, the rate of EF2-independent (spontaneous) translocation was increased in the absence of L41.