Biosynthesis of slaframine, (1S,6S,8aS)-1-acetoxy-6-aminooctahydroindolizine, a parasympathomimetic alkaloid of fungal origin. 3. Origin of the pyrrolidine ring.

The phytopathogen Rhizoctonia leguminicola has previously been shown to incorporate pipecolic acid into the piperidine alkaloids 1-acetoxy-6-aminooctahydroindolizine (slaframine) and 3,4,5-trihydroxyoctahydro-1-pyrindine. In the experiments described here, resting cultures of R. leguminicola were incubated with [1-14C]- and [2-14C]malonic acid and with [1-14C]- and [2-2H]acetic acid. Both acids were incorporated into the ring systems of both alkaloids. Mass spectrometric analysis of 2H-enriched slaframine showed that the label resides in the five-membered ring and that the methyl carbon of acetate is joined to the carboxyl carbon of pipecolate. A pipecolate-dependent decarboxylation of [1-14C]malonate was demonstrated in cell-free extracts of R. leguminicola. The results account for previously unattributed carbons in the two alkaloids and suggest the formation of an eight-carbon intermediate common to both alkaloids by acylation of malonate with pipecolic acid.     

Biosynthesis of slaframine, (1S,6S,8aS)-1-acetoxy-6-aminooctahydroindolizine, a parasympathomimetic alkaloid of fungal origin. 4. Metabolic fate of ethyl pipecolylacetate, 1,3-dioxooctahydroindolizine, and 1-hydroxyoctahydroindolizine in Rhizoctonia leguminicola.

Known or suspected intermediates in the biosynthesis of slaframine and 3,4,5-trihydroxyoctahydro-1-pyrindine, piperidine alkaloids of the phytopathogenic fungus Rhizoctonia leguminicola, were prepared and tested for biological conversions. Ethyl pipecolylacetate, an analogue of the postulated condensation product of pipecolic and malonic acids (two previously identified alkaloid precursors), was insufficiently stable for feeding experiments. The lactam of pipecolylacetate, 1,3-dioxooctahydroindolizine, was degraded by the fungus without direct incorporation into alkaloids. The known slaframine precursor 1-hydroxyoctahydroindolizine was prepared by a novel route which permitted high levels of deuterium enrichment at C-1 and C-3. Mass spectrometric examination of the slaframine biosynthesized from cis- and trans-[1,3,3-2H]-1-hydroxyoctahydroindolizine strengthened arguments that 1-oxooctahydroindolizine is an intermediate in slaframine biogenesis.     

Nutrient overload, insulin resistance, and ribosomal protein S6 kinase 1, S6K1

Nutrient overload leads to obesity, insulin resistance, and often type 2 diabetes. Whereas increased fat intake is commonly cited as the major factor in diet-induced dysmetabolic states, increased protein consumption also contributes, through elevated circulating amino acids. Recent studies have revealed that ribosomal protein S6 kinase 1, S6K1, an effector of mTOR, is sensitive to both insulin and nutrients, including amino acids. Although S6K1 is an effector of growth, recent reports show that amino acids also negatively affect insulin signaling through mTOR/S6K1 phosphorylation of IRS1. Moreover, rather than signaling through the class 1 PI3K pathway, amino acids appear to mediate mTOR activation through class 3 PI3K, or hVps34. Consistent with this, infusion of amino acids into humans leads to S6K1 activation, inhibition of insulin-induced class 1 PI3K activation, and insulin resistance. Thus, S6K1 may mediate deleterious effects, like insulin resistance, and potentially type 2 diabetes in the face of nutrient excess.     

Synthesis of 1,2-oxygenated 6-arylfurofuran lignan: stereoselective synthesis of (1S,2S,5R,6S)-1-hydroxysamin

(1S,2S,5R,6S)-6-(3,4-Methylenedioxyphenyl)-3,7-dioxabicyclo[3.3.0]octan-1,2-diol ((+)-1-hydroxysamin 1) was synthesized, starting from olefin 8. Stereoselective alpha-hydroxylation was achieved after converting 8 to aldehyde 13. Resulting unstable alpha-hydroxy aldehyde 14 was then transformed to (+)-1-hydroxysamin (1). This is a new efficient synthetic route to 1,2-oxygenated 6-arylfurofuran lignans.     

A facile synthesis of (1S,5R,6S)-5-azido-6-benzyloxycyclohex-2-en-1-ol

A facile and short synthesis of (1S,5R,6S)-5-azido-6-benzyloxycyclohex-2-en-1-ol (1) has been achieved in high yield starting from 4,5-epoxycyclohex-1-ene by using a catalytic asymmetric allylic oxidation reaction.     

Features of fibronectin-dependent activation of ribosomal protein S6 kinase (S6K1 and S6K2)

Integrin family of adhesion receptors play an important role in organizing the actin cytoskeleton and in signal transduction from the extracellular matrix. The previous studies have shown that exposure of fibroblast cells to extracellular matrix proteins activates ribosomal S6 kinase 1 (S6K1) pathway in a ligand dependent manner. Recently, a new, highly homologous ribosomal S6 kinase, termed S6K2, was identified. It has 70% amino acid identity in the overall sequence with S6K1, and the potential phosphorylation sites of S6K1 are conserved in S6K2. However, the N- and C-terminal domains of S6K2 are quite different from those of S6K1. In this study we have examined dynamics of fibronectin-induced activation of these two kinases, transiently expressed in human HEK 293 cells. Differences between profiles of activation of S6K1 and S6K2 were observed in the early period of fibronectin stimulation. Fibronectin-induced changes in S6K2 activity were closely correlated with phosphorylation at Ser423, which is homologues to Ser 434 of S6K1. Although we didn't observe considerable changes in phosphorylation of S6K1 at Ser434, suggesting potential differences in the regulation of these homologous kinases upon fibronectin stimulation.     

Salivary syndrome in horses: identification of slaframine in red clover hay.

An outbreak of salivary syndrome in horses in North Carolina was investigated. Rhizoctonia leguminicola was the predominant fungus isolated from toxic red clover hay. The fungus was less prevalent in the hay after 10 months of storage, and the hay had also decreased in biological activity after 10 months. Toxic hay caused extreme salivation, piloerection, respiratory distress, and increased frequency of defecation when fed to guinea pigs, and purified extracts of toxic hay and pure slaframine elicited these same responses when injected intraperitoneally into guinea pigs. The freshly acquired hay, based on the biological (slobber-producing) activity in hay and in purified extracts, contained the equivalent to 50 to 100 ppm (50 to 100 microgram/g) of slaframine, but this level had decreased after 10 months by about 10-fold to about 7 ppm. Slaframine and seven synthetic derivates of slaframine were used in presumptive gas-liquid chromatographic identification of this mycotoxin. Slaframine (1-acetoxy-6-amino-octahydroindolizine) was identified in purified extracts of toxic hay by gas-liquid chromatography-mass spectrometry after preparative thin-layer chromatography. This was the first direct identification of slaframine in toxic red clove hay.     

Synthesis and characterization of 6-O-beta-lactosyl-alpha,beta-lactoses, 1-O-(6-O-beta-lactosyl-beta-lactosyl)-(R,S)-glycerols, and 4,6-di-O-beta-D-galactopyranosyl-alpha,beta-D-glucoses.

1,2,3,2',3',4',6'-Hepta-O-acetyl-beta-lactose (4) was coupled with 2,3,6,2',3',4',6'-hepta-O-acetyl-alpha-lactosyl bromide (7) in the presence of Hg(CN)2 to afford 1,2,3,2',3',4',6'-hepta-O-acetyl-6-O-(2,3,6,2',3',4',6'-hepta-O-acetyl-b eta- lactosyl)-beta-lactose (11) which, upon O-deacetylation, gave 6-O-beta-lactosyl-alpha,beta-lactoses (64% from 4). In contrast, the reaction of 7 with benzyl 2,3,2',3',4',6'-hexa-O-acetyl-beta-lactoside in the presence of Hg(CN)2 produced 3,6,2',3',4',6'-hexa-O-acetyl-1,2-O- (2,3,2',3',4',6'-hexa-O-acetyl-1-O-benzyl-beta-lactos-6-yl orthoacetyl)-alpha-lactose (63%) and 3,6,2',3',4',6'-hexa-O-acetyl-1,2-O-(1- cyanoethylidene)-alpha-lactose (27%). The glycosidation of 4 using 2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl bromide in the presence of Hg(CN)2 afforded, after deprotection, 4,6-di-O-beta-D-galactopyranosyl-alpha,beta-D-glucoses (66%). The reaction of 11 with 1,2-di-O-benzyl-(R,S)-glycerols and trimethylsilyl trifluoromethanesulfonate yielded, after deprotection, 1-O-(6-O-beta-lactosyl-beta-lactosyl)-(R,S)-glycerols (18%). Under the same coupling conditions 11 reacted with 2-O-benzylglycerol to form 3-O-acetyl-2-O-benzyl-1-O-[2',3',4',6'-hexa-O-acetyl-6-O-(2,3,6,2',3',4' ,6'- hepta-O-acetyl-beta-lactosyl)-beta-lactosyl]-(R,S)-glycerols (16%).     

Phosphorylation and degradation of S6K1 (p70S6K1) in response to persistent JNK1 Activation

S6K (ribosomal S6 kinase p70, p70S6K) activation requires phosphorylation at two stages. The first phosphorylation is independent of insulin stimulation and mediated by an unknown kinase. The second phosphorylation is mediated by mTOR in insulin dependent manner. In this study, we identified JNK1 (c-Jun N-terminal kinase 1) as a kinase in the first phosphorylation. S6K protein was phosphorylated by JNK1 at S411 and S424 in the carboxyl terminal autoinhibitory domain. The phosphorylation was observed in kinase assay with purified S6K as a substrate, and in cells after JNK1 activation by TNF-α or MEKK1 expression. The phosphorylation was detected in JNK2 null cells, but not in JNK1 null cells after TNF-α treatment. When JNK1 activation was inhibited by MKK7 knockdown, the phosphorylation was blocked in cells. The phosphorylation led to S6K protein degradation in NF-κB deficient cells. The degradation was blocked by inhibition of proteasome activity with MG132. In wide type cells, the phosphorylation did not promote S6K degradation when IKK2 (IKKβ, IκB kinase beta) was activated. Instead, the phosphorylation allowed S6K activation by mTOR, which stabilizes S6K protein. In IKK2 null cells or cells treated by IKK2 inhibitor, the phosphorylation led to S6K degradation. These data suggest that S6K is phosphorylated by JNK1 and the phosphorylation makes S6K protein unstable in the absence of IKK2 activation. This study provides a mechanism for regulation of S6K protein stability.     

Interaction of ribosomal proteins S5, S6, S11, S12, S18 and S21 with 16 S rRNA

We have examined the effects of assembly of ribosomal proteins S5, S6, S11, S12, S18 and S21 on the reactivities of residues in 16 S rRNA towards chemical probes. The results show that S6, S18 and S11 interact with the 690-720 and 790 loop regions of 16 S rRNA in a highly co-operative manner, that is consistent with the previously defined assembly map relationships among these proteins. The results also indicate that these proteins, one of which (S18) has previously been implicated as a component of the ribosomal P-site, interact with residues near some of the recently defined P-site (class II tRNA protection) nucleotides in 16 S rRNA. In addition, assembly of protein S12 has been found to result in the protection of residues in both the 530 stem/loop and the 900 stem regions; the latter group is closely juxtaposed to a segment of 16 S rRNA recently shown to be protected from chemical probes by streptomycin. Interestingly, both S5 and S12 appear to protect, to differing degrees, a well-defined set of residues in the 900 stem/loop and 5'-terminal regions. These observations are discussed in terms of the effects of S5 and S12 on streptomycin binding, and in terms of the class III tRNA protection found in the 900 stem of 16 S rRNA. Altogether these results show that many of the small subunit proteins, which have previously been shown to be functionally important, appear to be associated with functionally implicated segments of 16 S rRNA.     

Hydrogen peroxide mediates Rac1 activation of S6K1

We previously reported that hydrogen peroxide (H2O2) mediates mitogen activation of ribosomal protein S6 kinase 1 (S6K1) which plays an important role in cell proliferation and growth. In this study, we investigated a possible role of H2O2 as a molecular linker in Rac1 activation of S6K1. Overexpression of recombinant catalase in NIH-3T3 cells led to the drastic inhibition of H2O2 production by PDGF, which was accompanied by a decrease in S6K1 activity. Similarly, PDGF activation of S6K1 was significantly inhibited by transient transfection or stable transfection of the cells with a dominant-negative Rac1 (Rac1N17), while overexpression of constitutively active Rac1 (Rac1V12) in the cells led to an increase in basal activity of S6K1. In addition, stable transfection of Rat2 cells with Rac1N17 dramatically attenuated the H2O2 production by PDGF as compared with that in the control cells. In contrast, Rat2 cells stably transfected with Rac1V12 produced high level of H2O2 in the absence of PDGF, comparable to that in the control cells stimulated with PDGF. More importantly, elimination of H2O2 produced in Rat2 cells overexpressing Rac1V12 inhibited the Rac1V12 activation of S6K1, indicating the possible role of H2O2 as a mediator in the activation of S6K1 by Rac1. However, H2O2 could be also produced via other pathway, which is independent of Rac1 or PI3K, because in Rat2 cells stably transfected with Rac1N17, H2O2 could be produced by arsenite, which has been shown to be a stimulator of H2O2 production. Taken together, these results suggest that H2O2 plays a pivotal role as a mediator in Rac1 activation of S6K1.     

Sequencing and characterization of telomere and subtelomere regions on rice chromosomes 1S, 2S, 2L, 6L, 7S, 7L and 8S

Telomeres, which are important for chromosome maintenance, are composed of long, repetitive DNA sequences associated with a variety of telomere-binding proteins. We characterized the organization and structure of rice telomeres and adjacent subtelomere regions on the basis of cytogenetic and sequence analyses. The length of the rice telomeres ranged from 5.1 to 10.8 kb, as revealed by both fibre-fluorescent in situ hybridization and terminal restriction-fragment assay. Physical maps of the chromosomal ends were constructed from a fosmid library. This facilitated sequencing of the telomere regions of chromosomes 1S, 2S, 2L, 6L, 7S, 7L and 8S. The resulting sequences contained conserved TTTAGGG telomere repeats, which indicates that the physical maps partly covered the telomere regions of the respective chromosome arms. These repeats were organized in the order of 5'-TTTAGGG-3' from the chromosome-specific region, except in chromosome 7S, in which seven inverted copies also existed in tandem array. Analysis of the telomere-flanking regions revealed the occurrence of deletions, insertions, or chromosome-specific substitutions of single nucleotides within the repeat sequences at the junction between the telomere and subtelomere. The sequences of the 500-kb regions of the seven chromosome ends were analysed in detail. A total of 598 genes were predicted in the telomeric regions. In addition, repetitive sequences derived from various kinds of retrotransposon were identified. No significant evidence for segmental duplication could be detected within or among the subtelomere regions. These results indicate that the rice chromosome ends are heterogeneous in both sequence and characterization.     

Mitogen-activated 70K S6 kinase. Identification of in vitro 40 S ribosomal S6 phosphorylation sites

Recently we purified and cloned the mitogen/oncogene-activated Mr 70,000 (70K) S6 kinase from the livers of rats treated with cycloheximide (Kozma, S. C., Lane, H. A., Ferrari, S., Luther, H., Siegmann, M., and Thomas, G. (1989) EMBO J. 8, 4125-4132; Kozma, S. C., Ferrari, S., Bassand, P., Siegmann, M., Totty, N., and Thomas, G. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 7365-7369). Prior to determining the ability of this kinase to phosphorylate the same sites observed in S6 in vivo, we established the effects of different cations and autophosphorylation on kinase activity. The results show that the 70K S6 kinase is dependent on Mg2+ for activity and that this requirement cannot be substituted for by Mn2+. Furthermore, 50-fold lower concentrations of Mn2+ block the effect of Mg2+ on the kinase. This effect is not limited to Mn2+ but can be substituted for by a number of cations, with Zn2+ being the most potent inhibitor, IC50 approximately 2 microM. In the presence of optimum Mg2+ concentrations the enzyme incorporates an average of 1.2 mol of phosphate/mol of kinase and an average of 3.7 mol of phosphate/mol of S6. The autophosphorylation reaction appears to be intramolecular and leads to a 25% reduction in kinase activity toward S6. In the case of S6 all of the sites of phosphorylation are found to reside in a 19-amino acid peptide at the carboxyl end of the protein. Four of these sites have been identified as Ser235, Ser236, Ser240, and Ser244, equivalent to four of the five sites previously observed in vivo (Krieg, J., Hofsteenge, J., and Thomas, G. (1988) J. Biol. Chem. 263, 11473-11477). A fifth mole of phosphate is incorporated at low stoichiometry into the peptide, but the amino acid which is phosphorylated cannot be unequivocally assigned. The low level of phosphorylation of the fifth site in vitro is discussed with regard to known results and to a potential three-dimensional model for the carboxyl terminus of S6.     

Expression, purification, and characterization of a structurally disordered and functional C-terminal autoinhibitory domain (AID) of the 70 kDa 40S ribosomal protein S6 kinase-1 (S6K1)

S6K1 is a member of the AGC subfamily of serine-threonine protein kinases, whereby catalytic activation requires dual phosphorylation of critical residues in the conserved T-loop (T229) and hydrophobic motif (HM; T389) peptide regions of its catalytic kinase domain (residues 1-398). In addition to its kinase domain, S6K1 contains a C-terminal autoinhibitory domain (AID; residues 399-502), which prevents T-loop and HM phosphorylation; and autoinhibition is relieved on multi-site Ser-Thr phosphorylation of the AID (S411, S418, T421, and S424). Interestingly, 66 of the 104 C-terminal AID amino acid residues were computer predicted to exist in structurally disordered peptide regions, begetting interest as to how such dynamics could be coupled to autoregulation. To begin addressing this issue, we developed and optimized protocols for efficient AID expression and purification. Consistent with computer predictions, aberrant mobilities in both SDS-PAGE and size-exclusion chromatography, as well as low chemical shift dispersion in (1)H-(15)N HSQC NMR spectra, indicated purified recombinant AID to be largely unfolded. Yet, trans-addition of purified AID effectively inhibited PDK1-catalyzed T-loop phosphorylation of a catalytic kinase domain construct of S6K1. Using an identical purification protocol, similar protein yields of a tetraphospho-mimic mutant AID(D(2)ED) construct were obtained; and this construct displayed only weak inhibition of PDK1-catalyzed T229 phosphorylation. Purification of the structurally 'disordered' and functional C-terminal AID and AID(D(2)ED) constructs will facilitate studies aimed to understand the role of conformational plasticity and protein phosphorylation in modulating autoregulatory domain-domain interactions.     

Mitogen-independent phosphorylation of S6K1 and decreased ribosomal S6 phosphorylation in senescent human fibroblasts

The p70 ribosomal S6 kinase (S6K1) is rapidly activated following growth factor stimulation of quiescent fibroblasts and inhibition of this enzyme results in a G(1) arrest. Phosphorylation of the ribosomal S6 protein by S6K1 regulates the translation of both ribosomal proteins and initiation factors, leading to an increase in protein synthesis. We have examined the activation of S6K1 in human fibroblasts following mitogen stimulation. In early passage fibroblasts S6K1 is activated following serum stimulation as evidenced by increased kinase activity and site-specific phosphorylation. In contrast, site-specific phosphorylation of S6K1 at Thr421/Ser424 is diminished in senescent fibroblast cultures. A second phosphorylation site within S6K1 (Ser411) is phosphorylated even in the absence of serum stimulation and the enzyme shows increased phosphorylation as judged by decreased electrophoretic mobility. Inhibitor studies indicate that this phosphorylation is dependent upon the mammalian target of rapamycin, PI 3-kinase, and the MAPK pathway. In order to understand the consequences of the altered phosphorylation of the S6K1, we examined the phosphorylation state of the ribosomal S6 protein. In early passage fibroblasts the ribosomal S6 protein is phosphorylated upon serum stimulation while the phosphorylation of the ribosomal S6 protein is drastically reduced in senescent fibroblasts. These results suggest that the intracellular regulators of S6K1 are altered during replicative senescence leading to a deregulation of the enzyme and a loss of ribosomal S6 phosphorylation.