Involvement of leucine residues at positions 107, 112, and 115 in a leucine-rich repeat motif of human Toll-like receptor 2 in the recognition of diacylated lipoproteins and lipopeptides and Staphylococcus aureus peptidoglycans

S-(2,3-bispalmitoyloxypropyl)Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe (FSL-1) derived from Mycoplasma salivarium stimulated NF-kappaB reporter activity in human embryonic kidney 293 (HEK293) cells transfected with Toll-like receptor 2 (TLR2) or cotransfected with TLR2 and TLR6, but not in HEK293 cells transfected with TLR6, in a dose-dependent manner. The activity was significantly higher in HEK293 cells transfected with both TLR2 and TLR6 than in HEK293 cells transfected with only TLR2. The deletion mutant TLR2(DeltaS40-I64) (a TLR2 mutant with a deletion of the region of Ser(40) to Ile(64)) failed to activate NF-kappaB in response to FSL-1. The deletion mutant TLR2(DeltaC30-S39) induced NF-kappaB reporter activity, but the level of activity was significantly reduced compared with that induced by wild-type TLR2. A TLR2 point mutant with a substitution of Glu(178) to Ala (TLR2(E178A)), TLR2(E180A), TLR2(E190A), and TLR2(L132E) induced NF-kappaB activation when stimulated with FSL-1, M. salivarium lipoproteins, and Staphylococcus aureus peptidoglycans, but TLR2(L107E), TLR2(L112E) (a TLR2 point mutant with a substitution of Leu(112) to Glu), and TLR2(L115E) failed to induce NF-kappaB activation, suggesting that these residues are essential for their signaling. Flow cytometric analysis demonstrated that TLR2(L115E), TLR2(L112E), and TLR2(DeltaS40-I64) were expressed on the cell surface of the transfectants as wild-type TLR2 and TLR2(E190A) were. In addition, these mutants, except for TLR2(E180A), functioned as dominant negative form of TLR2. This study strongly suggested that the extracellular region of Ser(40)-Ile(64) and leucine residues at positions 107, 112, and 115 in a leucine-rich repeat motif of TLR2 are involved in the recognition of mycoplasmal diacylated lipoproteins and lipopeptides and in the recognition of S. aureus peptidoglycans.     

Stat5B shuttles between cytoplasm and nucleus in a cytokine-dependent and -independent manner

In response to cytokine stimuli, Stats are phosphorylated and translocated to the nucleus to activate target genes. Then, most are dephosphorylated and returned to the cytoplasm. Using Ba/F3 cells, we found that the nuclear export of Stat5B by cytokine depletion was inhibited by leptomycin B (LMB), a specific inhibitor of nuclear export receptor chromosome region maintenance 1. Interestingly, LMB treatment in the absence of cytokine led to the accumulation of Stat5B in the nucleus, suggesting that Stat5B shuttles between the nucleus and the cytoplasm as a monomer without cytokine stimulation. This notion is supported by the observation that LMB-induced accumulation of Stat5B in the nucleus was also observed with Stat5B having a mutated tyrosine 699, which is essential for dimer formation. Using a series of mutant Stat5Bs, we identified a part of the coiled coil domain to be a critical region for monomer nuclear import and a more N-terminal region to be critical for the cytokine stimulation dependent import of Stat5B. Taken together, we propose a model in which Stat5B shuttles between the nucleus and cytoplasm by two different mechanisms, one being a factor-independent constitutive shuttling by monomeric form, and the other, a factor stimulation-dependent one regulated by tyrosine phosphorylation and subsequent dimerization.     

Indole alkaloids from Rauvolfia bahiensis A.DC. (Apocynaceae)

Four indole alkaloids, 12-methoxy-N(a)-methyl-vellosimine, demethoxypurpeline, 12-methoxyaffinisine, and 12-methoxy-vellosimine, in addition to picrinine, vinorine, raucaffrinoline, normacusine B, norseredamine, seredamine, 10-methoxynormacusine B, norpurpeline and purpeline, were isolated from the bark or leaf extracts of Rauvolfia bahiensis.     

Time-Measuring Processes Involved in the Photoperiodic Response of Lemna paucicostata 441

Lemna paucicostata 441 exposed to a single dark period of variouslengths showed a rhythmic flowering response with a 22- to 24-hperiod, even when the dark period was preceded by continuouslight. The critical night length (about 12 h) was scarcely influencedby pretreatment with 8D–4L (8 h of darkness followed by4 h of light), 8D–8L or 8D–12L. However, the rhythmof the response in the second cycle was markedly damped by thepretreatment with 8D–4L or 8D–12L, and was slightlyamplified by 8D–8L. The flowering response to a red-light interruption given atdifferent times in the inductive dark period also showed circadianrhythmicity even when the dark period was preceded by continuouslight, and this rhythmicity was scarcely influenced by a dark-lighttreatment given prior to the inductive dark period. A red-lightinterruption given at the 6th or 14th hour of the dark periodmarkedly shifted the phase of the rhythm of the response tothe length of the following dark period (the former delayedand the latter advanced), but that given at the same phase markedlyweakened and disturbed the rhythmicity of the response to ared-light interruption given in the following dark period. (Received March 21, 1992; Accepted June 12, 1992)     

Eukaryotic-like protein serine/threonine kinases in Myxococcus xanthus,a developmental bacterium exhibiting social behavior

Myxococcus xanthus, a gram-negative bacterium exhibits a spectacular life cycle and social behavior. Its developmental cycle and multicellular morphogenesis resemble those of eukaryotic slime molds such as Dictyostelium discoideum. On the basis of this resemblance, we explored the existence of eukaryotic-like protein serine/threonine kinases which are known to play important roles in signal transduction during development of D. discoideum. It was indeed found that M. xanthus contains a large family of protein serine/threonine kinases related to the eukaryotic enzymes. This is the first unambiguous demonstration of eukaryotic-like protein serine/threonine kinases in the prokaryotes. © 1993 Wiley-Liss, Inc.     

Determination of rhoifolin and daidzin in human plasma by high-performance liquid chromatography

A method for determining flavonoids in human plasma is presented for application to pharmacokinetic studies of two flavonoids, rhoifolin and daidzin. Isocratic reversed-phase high-performance liquid chromatography (HPLC) was used with genistin as an internal standard and solid-phase extraction using a Sep-Pak C₁₈ cartridge. The mobile phases were acetonitrile—0.1 M ammonium acetate solution (20:80, v/v) for rhoifolin and methanol—0.1 M ammonium acetate solution (33:67, v/v) for daidzin. The detection limits on-column were 2 ng for rhoifolin and 0.5 ng for daidzin.