Oleanolic glycosides from Pometia ridleyi

Six triterpenoid saponins were isolated from the stem bark of Pometia ridleyi along with two known saponins, acutoside A and calenduloside C. Their structures were established using one- and two-dimensional NMR and mass spectrometry as 3-O-beta-D-apiofuranosyl-(1-->3)-[beta-D-glucopyranosyl-(1-->2)]-beta-D-glucopyranosyl-, 3-O-beta-D-apiofuranosyl-(1-->3)-alpha-L-arabinopyranosyl-(1-->3)-[beta-D-glucopyranosyl-(1-->2)]-beta-D-glucopyranosyl-, 3-O-beta-D-apiofuranosyl-(1-->3)-beta-D-galactopyranosyl-(1-->3)-[beta-D-glucopyranosyl-(1-->2)]-beta-D-glucopyranosyl-, 3-O-alpha-L-arabinopyranosyl-(1-->3)-[beta-D-glucopyranosyl-(1-->2)]-alpha-L-arabinopyranosyl-, 3-O-beta-D-galactopyranosyl-(1-->3)-[beta-D-glucopyranosyl-(1-->2)]-alpha-L-arabinopyranosyl-, 3-O-beta-D-apiofuranosyl-(1-->3)-beta-D-galactopyranosyl-(1-->3)-[beta-D-glucopyranosyl-(1-->2)]-alpha-L-arabinopyranosyl-oleanolic acid. The EtOH and EtOAc extracts of the stem bark showed no cytotoxic activity. At a concentration of 23 microg/ml, the saponin mixture showed haemolytic activity and caused 50% haemolysis of a 10% suspension of sheep erythrocytes.     

Susceptibility and adaptive response to bile salts in Propionibacterium freudenreichii: physiological and proteomic analysis

Tolerance to digestive stresses is one of the main factors limiting the use of microorganisms as live probiotic agents. Susceptibility to bile salts and tolerance acquisition in the probiotic strain Propionibacterium freudenreichii SI41 were characterized. We showed that pretreatment with a moderate concentration of bile salts (0.2 g/liter) greatly increased its survival during a subsequent lethal challenge (1.0 g/liter, 60 s). Bile salts challenge led to drastic morphological changes, consistent with intracellular material leakage, for nonadapted cells but not for preexposed ones. Moreover, the physiological state of the cells during lethal treatment played an important role in the response to bile salts, as stationary-phase bacteria appeared much less sensitive than exponentially growing cells. Either thermal or detergent pretreatment conferred significantly increased protection toward bile salts challenge. In contrast, some other heterologous pretreatments (hypothermic and hyperosmotic) had no effect on tolerance to bile salts, while acid pretreatment even might have sensitized the cells. Two-dimensional electrophoresis experiments revealed that at least 24 proteins were induced during bile salts adaptation. Identification of these polypeptides suggested that the bile salts stress response involves signal sensing and transduction, a general stress response (also triggered by thermal denaturation, oxidative toxicity, and DNA damage), and an alternative sigma factor. Taken together, our results provide new insights into the tolerance of P. freudenreichii to bile salts, which must be taken into consideration for the use of probiotic strains and the improvement of technological processes.     

Autophosphorylation of the catalytic subunit of the DNA-dependent protein kinase is required for efficient end processing during DNA double-strand break repair

The DNA-dependent protein kinase (DNA-PK) plays an essential role in nonhomologous DNA end joining (NHEJ) by initially recognizing and binding to DNA breaks. We have shown that in vitro, purified DNA-PK undergoes autophosphorylation, resulting in loss of activity and disassembly of the kinase complex. Thus, we have suggested that autophosphorylation of the DNA-PK catalytic subunit (DNA-PKcs) may be critical for subsequent steps in DNA repair. Recently, we defined seven autophosphorylation sites within DNA-PKcs. Six of these are tightly clustered within 38 residues of the 4,127-residue protein. Here, we show that while phosphorylation at any single site within the major cluster is not critical for DNA-PK's function in vivo, mutation of several sites abolishes the ability of DNA-PK to function in NHEJ. This is not due to general defects in DNA-PK activity, as studies of the mutant protein indicate that its kinase activity and ability to form a complex with DNA-bound Ku remain largely unchanged. However, analysis of rare coding joints and ends demonstrates that nucleolytic end processing is dramatically reduced in joints mediated by the mutant DNA-PKcs. We therefore suggest that autophosphorylation within the major cluster mediates a conformational change in the DNA-PK complex that is critical for DNA end processing. However, autophosphorylation at these sites may not be sufficient for kinase disassembly.     

Detailed glycan analysis of serum glycoproteins of patients with congenital disorders of glycosylation indicates the specific defective glycan processing step and provides an insight into pathogenesis

The fundamental importance of correct protein glycosylation is abundantly clear in a group of diseases known as congenital disorders of glycosylation (CDGs). In these diseases, many biological functions are compromised, giving rise to a wide range of severe clinical conditions. By performing detailed analyses of the total serum glycoproteins as well as isolated transferrin and IgG, we have directly correlated aberrant glycosylation with a faulty glycosylation processing step. In one patient the complete absence of complex type sugars was consistent with ablation of GlcNAcTase II activity. In another CDG type II patient, the identification of specific hybrid sugars suggested that the defective processing step was cell type-specific and involved the mannosidase III pathway. In each case, complementary serum proteome analyses revealed significant changes in some 31 glycoproteins, including components of the complement system. This biochemical approach to charting diseases that involve alterations in glycan processing provides a rapid indicator of the nature, severity, and cell type specificity of the suboptimal glycan processing steps; allows links to genetic mutations; indicates the expression levels of proteins; and gives insight into the pathways affected in the disease process.     

Purification of a nitrate reductase kinase from Spinacea oleracea leaves, and its identification as a calmodulin-domain protein kinase

Spinach (Spinacea oleracea L.) nitrate reductase (NR) is inactivated by phosphorylation on serine-543, followed by binding of the phosphorylated enzyme to 14-3-3 proteins. We purified one of several chromatographically distinct NR_serine-543 kinases from spinach leaf extracts, and established by Edman sequencing of 80 amino acid residues that it is a calcium-dependent (calmodulin-domain) protein kinase (CDPK), with peptide sequences very similar to Arabidopsis CDPK6 (accession no. U20623; also known as CPK3). The spinach CDPK was recognized by antibodies raised against Arabidopsis CDPK. Nitrate reductase was phosphorylated at serine-543 by bacterially expressed His-tagged CDPK6, and the phosphorylated NR was inhibited by 14-3-3 proteins. However, the bacterially expressed CDPK6 had a specific activity approx. 200-fold lower than that of the purified spinach enzyme. The physiological control of NR by CDPK is discussed, and the regulatory properties of the purified CDPK are considered with reference to current models for reversible intramolecular binding of the calmodulin-like domain to the autoinhibitory junction of CDPKs. Received: 12 February 1998 / Accepted: 28 May 1998     

Functional Promiscuity of Gene Regulation by Serpentine Receptors in Dictyostelium discoideum

Serpentine receptors such as smoothened and frizzled play important roles in cell fate determination during animal development. In Dictyostelium discoideum, four serpentine cyclic AMP (cAMP) receptors (cARs) regulate expression of multiple classes of developmental genes. To understand their function, it is essential to know whether each cAR is coupled to a specific gene regulatory pathway or whether specificity results from the different developmental regulation of individual cARs. To distinguish between these possibilities, we measured gene induction in car1 car3 double mutant cell lines that express equal levels of either cAR1, cAR2, or cAR3 under a constitutive promoter. We found that all cARs efficiently mediate both aggregative gene induction by cAMP pulses and induction of postaggregative and prespore genes by persistent cAMP stimulation. Two exceptions to this functional promiscuity were observed. (i) Only cAR1 can mediate adenosine inhibition of cAMP-induced prespore gene expression, a phenomenon that was found earlier in wild-type cells. cAR1’s mediation of adenosine inhibition suggests that cAR1 normally mediates prespore gene induction. (ii) Only cAR2 allows entry into the prestalk pathway. Prestalk gene expression is induced by differentiation-inducing factor (DIF) but only after cells have been prestimulated with cAMP. We found that DIF-induced prestalk gene expression is 10 times higher in constitutive cAR2 expressors than in constitutive cAR1 or cAR3 expressors (which still have endogenous cAR2), suggesting that cAR2 mediates induction of DIF competence. Since in wild-type slugs cAR2 is expressed only in anterior cells, this could explain the so far puzzling observations that prestalk cells differentiate at the anterior region but that DIF levels are actually higher at the posterior region. After the initial induction of DIF competence, cAMP becomes a repressor of prestalk gene expression. This function can again be mediated by cAR1, cAR2, and cAR3.Recent years have seen the discovery of critical roles in animal development for serpentine receptors, which are usually coupled to heterotrimeric G proteins. The insect sigaling peptides hedgehog and wingless and their mammalian counterparts sonic hedgehog, desert hedgehog, and indian hedgehog and the wnt factors control a multitude of inductive events during all stages of embryogenesis. The hedgehog signal is detected by two different serpentine receptors, smoothened (1, 40) and patched (21, 38), whereas the wingless or wnt signal is detected by the serpentine receptor D-frizzled-2 (3). In the social amoeba Dictyostelium discoideum, serpentine cyclic AMP (cAMP) receptors (cARs) control induction of cell differentiation during the entire course of development. Starving cells secrete cAMP pulses that induce chemotaxis and expression of genes required for the aggregation process. Cells aggregate to form mounds, which ultimately transform into fruiting structures that consist of a globular spore mass supported by a column of stalk cells. cAMP induces entry into the spore differentiation pathway as well as synthesis of a lipophilic factor, differentiation-inducing factor (DIF), which induces entry into the stalk differentiation pathway (see reference 5). At an early stage of development cAMP synergizes with DIF to induce prestalk genes, but later it becomes an inhibitor of stalk gene expression (2). cARs were shown previously to mediate induction of aggregative genes by cAMP pulses (20) as well as cAMP induction of prespore genes and repression of prestalk genes (31, 37). Remarkably, the target for the latter critical step in cell fate determination is glycogen synthase kinase 3 (GSK-3), a zeste white-3 homolog, which is the target for the effects of wingless and wnt in insects and vertebrates, respectively (7, 34).Four cARs, showing 54 to 69% amino acid identity, are expressed in a stage- and cell-type-specific manner. cAR1 is predominantly expressed before and during aggregation (18). cAR3 is expressed at late aggregation, and expression is later restricted to the prespore cell population (13, 44). cAR2 and cAR4 are both expressed exclusively in the prestalk cell population after aggregation (19, 30). cAR knockout cell lines were generated to examine the role of the individual cARs in Dictyostelium development. car1 null cells neither aggregate nor express developmental genes but can be triggered to express aggregative and postaggregative genes by stimulation with cAMP (37, 39). car3 null cells aggregate and develop normally (13). car1 car3 double gene disruptants do not aggregate, and developmental gene expression cannot be restored with cAMP, indicating that cAR1 or cAR3 shows functional redundancy and that either one or the other has to be present for gene induction to occur (10, 36). car2 null cells are blocked in the mound stage, while car4 null cells show abnormal slug morphogenesis and culmination. Both lines show reduced expression of prestalk genes and enhanced expression of prespore genes (19, 29).To understand the function of the four cARs, it is essential to know whether each receptor is coupled to a specific signal transduction pathway that controls a specific cell differentiation event or whether each receptor can activate multiple cell differentiation pathways. In the latter case, it is not the presence of a specific receptor that determines whether a response occurs but the availability of the downstream signaling pathway. To determine whether individual receptors have unique functions in developmental gene expression, we examined gene regulation in cell lines that display about equal levels of cAR1, cAR2, and cAR3 in a car1 car3 mutant background. Our results show that with two exceptions, all three receptors can transduce both the excitation and adaptation components of the different cAMP-regulated gene induction events with almost equal levels of efficiency.     

Quercitol links the physiology, taxonomy and evolution of 279 eucalypt species

Aim  Increasing aridity over geological time-scales has driven a high degree of speciation within the Eucalyptus group in Australia. Isolation of gene pools by climatic and edaphic conditions and high rates of out-crossing have given rise to a large diversity of adaptive traits. Among these traits, adaptations of cellular biochemistry are likely to be significant in preserving cellular function during arid conditions. The aim of this study was to determine the quantitative and qualitative distribution of soluble carbohydrates and polyols in Eucalyptus .
Location  Australia.
Methods  We sampled 279 of the 700+ documented eucalypts (in the three genera comprising the eucalypts: Angophora Cav., Corymbia Hill & Johnson and Eucalyptus L'Hér.) and analysed leaf tissues for the occurrence of low-molecular-weight carbohydrates and polyols.
Results  We have uncovered a discrete pattern in concentration of quercitol (a cyclitol) that correlates strongly with the current taxonomic classification based on both morphology and DNA sequencing. We also uncovered a further and stronger correlation between the presence of quercitol in leaf tissues and a reduced growth (mallee) form.
Main conclusions  These findings, together with the chemical properties of quercitol, suggest that we have uncovered a chemical marker of structural adaptations to arid conditions, thus providing a putative, broad-scale functional link to adaptation to aridity.     

Electrochemical regeneration of redox cofactors and mediators — The key to bioelectrosynthesis

Bioelectrosynthesis is a novel approach to developing new routes for the synthesis of fine chemicals and pharmaceuticals which combines the speed and selectivity of enzymic catalysis with an electrode as the ultimate source of reducing or oxidizing power. The electrochemistries of the NAD⁺ /NADH, FAD/FADH₂, and FMN/FMNH₂ systems are used to illustrate some basic principles. Technical and economic factors are also considered.