Eudesmanolides from Calea trichomata

Chemical analysis of Calea trichomata yielded, besides four known eudesmanolides, a new eudesmanolide, trichomatolide A. The structure of the new compound was established by chemical and spectral methods.     

Nucleotide binding to the multidrug resistance P-glycoprotein as studied by ESR spectroscopy

Electron spin resonance (ESR) spectroscopy using spin-labeled ATP was used to study nucleotide binding to and structural transitions within the multidrug resistance P-glycoprotein, P-gp. Spin-labeled ATP (SL-ATP) with the spin label attached to the ribose, was observed to be an excellent substrate analogue for P-gp. SL-ATP was hydrolyzed in a drug-stimulated fashion at about 14% of the rate for normal ATP and allowed reversible trapping of the enzyme in transition and ground states. Equilibrium binding of a total of two nucleotides per P-gp was observed with a binding affinity of 366 microM in the presence of Mg2+ but in the absence of transport substrates such as verapamil. Binding of SL-ATP to wild-type P-gp in the presence of verapamil resulted in reduction of the protein-bound spin-label moiety, most likely due to a conformational transition within P-gp that positioned cysteines in close proximity to the spin label to allow chemical reduction of the radical. We circumvented this problem by using a mutant of P-gp in which all naturally occurring cysteines were substituted for alanines. Equilibrium binding of SL-ATP to this mutant P-gp resulted in maximum binding of two nucleotides; the binding affinity was 223 microM in the absence and 180 microM in the presence of verapamil. The corresponding ESR spectra of wild-type and Cys-less P-gp in the presence of SL-ATP indicate that a cysteine side chain of P-gp is located close to the ribose of the bound nucleotide. Trapping SL-ATP as an AlF(x)-adduct resulted in ESR spectra that showed strong immobilization of the radical, supporting the formation of a closed conformation of P-gp in its transition state. This study is the first to employ ESR spectroscopy with the use of spin-labeled nucleotide analogues to study P-glycoprotein. The study shows that SL-ATP is an excellent substrate analogue that will allow further exploration of structure and dynamics within the nucleotide binding domains of this important enzyme.     

Mutational analysis of conserved carboxylate residues in the nucleotide binding sites of P-glycoprotein

Mutagenesis was used to investigate the functional role of six pairs of aspartate and glutamate residues (D450/D1093, E482/E1125, E552/E1197, D558/D1203, D592/D1237, and E604/E1249) that are highly conserved in the nucleotide binding sites of P-glycoprotein (Mdr3) and of other ABC transporters. Removal of the charge in E552Q/E1197Q and D558N/D1203N produced proteins with severely impaired biological activity when the proteins were analyzed in yeast cells for cellular resistance to FK506 and restoration of mating in a ste6Delta mutant. Mutations at other acidic residues had no apparent effect in the same assays. These four mutants were expressed in Pichia pastoris, purified to homogeneity, and biochemically characterized with respect to ATPase activity. Studies with purified proteins showed that mutants D558N and D1203N retained 14 and 30% of the drug-stimulated ATPase activity of wild-type (WT) Mdr3, respectively, and vanadate trapping of 8-azido[alpha-(32)P]nucleotide confirmed slower basal and drug-stimulated 8-azido-ATP hydrolysis compared to that for WT Mdr3. The E552Q and E1197Q mutants showed no drug-stimulated ATPase activity. Surprisingly, drugs did stimulate vanadate trapping of 8-azido[alpha-(32)P]nucleotide in E552Q and E1197Q at a level similar to that of WT Mdr3. This suggests that formation of the catalytic transition state can occur in these mutants, and that the bond between the beta- and gamma-phosphates is hydrolyzed. In addition, photolabeling by 8-azido[alpha-(32)P]nucleotide in the presence or absence of drug was also detected in the absence of vanadate in these mutants. These results suggest that steps after the transition state, possibly involved in release of MgADP, are severely impaired in these mutant enzymes.     

Increased SCF/c‐kit by hypoxia promotes autophagy of human placental chorionic plate‐derived mesenchymal stem cells via regulating the phosphorylation of mTOR

Hypoxia triggers physiological and pathological cellular processes, including proliferation, differentiation, and death, in several cell types. Mesenchymal stem cells (MSCs) derived from various tissues have self‐renewal activity and can differentiate towards multiple lineages. Recently, it has been reported that hypoxic conditions tip the balance between survival and death by hypoxia‐induced autophagy, although the underlying mechanism is not clear. The objectives of this study are to compare the effect of hypoxia on the self‐renewal of bone marrow‐derived mesenchymal stem cells (BM‐MSCs) and placental chorionic plate‐derived mesenchymal stem cells (CP‐MSCs) and to investigate the regulatory mechanisms of self‐renewal in each MSC type during hypoxia. The expression of self‐renewal markers (e.g., Oct4, Nanog, Sox2) was assessed in both cell lines. PI3K and stem cell factor (SCF) expression gradually increased in CP‐MSCs but were markedly downregulated in BM‐MSCs by hypoxia. The phosphorylation of ERK and mTOR was augmented by hypoxia in CP‐MSCs compared to control. Also, the expression of LC3 II, a component of the autophagosome and the hoof‐shaped autophagosome was detected more rapidly in CP‐MSCs than in BM‐MSCs under hypoxia. Hypoxia induced the expression of SCF in CP‐MSCs and increased SCF/c‐kit pathway promotes the self‐renewal activities of CP‐MSCs via an autocrine/paracrine mechanism that balances cell survival and cell death events by autophagy. These activities occur to a greater extent in CP‐MSCs than in BM‐MSCs through regulating the phosphorylation of mTOR. These findings will provide useful guidelines for better understanding the function of SCF/c‐kit in the self‐renewal and autophagy‐regulated mechanisms that promote of MSC survival. J. Cell. Biochem. 114: 79–88, 2012. © 2012 Wiley Periodicals, Inc.     

Distribution of TGF‐β isoforms and signaling intermediates in corneal fibrotic wound repair

In this study, temporal and spatial distribution of three TGF‐β isoforms and their downstream signaling pathways including pSmad2 and p38MAPK were examined during fibrotic wound repair. In normal chick corneas, TGF‐β1, ‐2, and ‐3 were weakly detected in Bowman's layer (BL). In healing corneas, TGF‐β1 was primarily deposited in the fibrin clot and the unwounded BL. TGF‐β2 was highly expressed in healing epithelial and endothelial cells, and numerous active fibroblasts/myofibroblasts. TGF‐β3 was mainly detected in the unwound region of basal epithelial cells. α‐Smooth muscle actin (α‐SMA) was initially appeared in the posterior region of repairing stroma at day 3, and was detected in the entire healing stroma by day 7. Notably, α‐SMA was absent in the central region of healing stroma by day 14, and its staining pattern was similar to those of TGF‐β2 and p38MAPK. By contrast, pSmad2 was mainly detected in the fibroblasts. In normal cornea, laminin was mainly detected in both epithelial basement membrane (BM) and Descemet's membrane (DM). By contrast to reconstitution of the BM in the wound region, the DM was not repaired although endothelial layer was regenerated, indicating that high levels of TGF‐β2 were released into the posterior region of healing stroma on day 14. High levels of α‐SMA staining, shown in cultured repair stromal cells from healing corneas on day 14 and in TGF‐β2 treated normal stromal cells, were significantly reduced by p38MAPK inhibition. Collectively, this study suggests that TGF‐β2‐mediated myofibroblast transformation is mediated, at least partly, by the p38MAPK pathway in vivo. J. Cell. Biochem. 108: 476–488, 2009. © 2009 Wiley‐Liss, Inc.     

Properties of P-glycoprotein with mutations in the "catalytic carboxylate" glutamate residues

It is known from earlier work that two conserved Glu residues, designated "catalytic carboxylates," are critical for function in P-glycoprotein (Pgp). Here the role of these residues (Glu-552 and Glu-1197 in mouse MDR3 Pgp) was studied further. Mutation E552Q or E1197Q reduced Pgp-ATPase to low but still measurable rates. Two explanations previously offered for effects of these mutations, namely that ADP release is slowed or that a second (drug site-resetting) round of ATP hydrolysis is blocked, were evaluated and appeared unsatisfactory. Thus the study was extended to include E552A, -D, and -K and E1197A, -D, and -K mutants. All reduced ATPase to similar low but measurable rates. Orthovanadate-trapping experiments showed that mutation to Gln, Ala, Asp, or Lys altered characteristics of the transition state but did not eliminate its formation in contrast e.g. with mutation of the analogous catalytic Glu in F1-ATPase. Retention of ATP as well as ADP was seen in Ala, Asp, and Lys mutants. Mutation E552A in nucleotide binding domain 1 (NBD1) was combined with mutation S528A or S1173A in the LSGGQ sequence of NBD1 or NBD2, respectively. Synergistic effects were seen. E552A/S1173A had extremely low turnover rate for ATPase, while E552A/S528A showed zero or close to zero ATPase. Both showed orthovanadate-independent retention of ATP and ADP. We propose that mutations of the catalytic Glu residues interfere with formation and characteristics of a closed conformation, involving an interdigitated NBD dimer interface, which normally occurs immediately following ATP binding and progresses to the transition state.     

Seasonal Rotifer Dynamics in the Long-term (1969–2002) Record from Lake Kinneret (Israel)

Long-term (1969–2002) data record of biomass distribution of rotifers in Lake Kinneret is combined with previously published information on their metabolic activity and newly calculated population dynamics parameters to synthesize a model of their seasonal dynamics in Lake Kinneret. Nineteen rotifer species were recorded in routine samples collected in Lake Kinneret (Israel) in 7 offshore (deeper than 5 m), stations, at 12 discrete depths during 1969–2002. Organisms were sorted and counted (including external egg carrying females), biomass was measured and calculated for the entire lake stock (g_w.w m⁻²; mg l⁻¹). Rates of grazing, respiration and production were measured experimentally at three different temperature ranges. Results were extrapolated to the lake community for months with similar temperatures. Rotifera comprised 7% of total zooplankton biomass in Lake Kinneret whilst Cladocera and Copepoda 58 and 35% respectively. Rotifers were found to be more abundant during December–June and decline in summer months. Monthly (1969–2001) means indicated total grazing capacity of rotifers as 11%, respiration as 9% and production as 3.7% of the total zooplankton metabolic activity. Positive relations were indicated between rotifer and small bodied cladoceran numerical concentrations. Population growth models suggest that rotifers are not food limited in Lake Kinneret but that fish predation plays an important role in regulating abundance in spring-summer and fall.     

The flavonoids of tetragonotheca (compositae)

Fifteen flavonols, five aglycones and ten glucosides were isolated from the four species of Tetragonotheca, T. repanda, T. helianthoides, T. texana and T. ludoviciana. Included among the isolated flavonols are four previously unreported 7-O-glucosides, 6-hydroxykaempferol 7-O-glucoside, 6-hydroxykaempferol 6-methyl ether 7-O-glucoside, quercetagetin 6,3′-dimethyl ether 7-O-glucoside and quercetagetin 3,6-dimethyl ether 7-O-glucoside.