Six new constituents from the roots of Erythrina variegata

Three new isoflavonoids, eryvarins M-O (1-3), two new 2-arylbenzofurans, eryvarins P and Q (4 and 5), and a new 3-aryl-2,3-dihydrobenzofuran, eryvarin R (6), together with three known compounds, were isolated from the roots of Erythrina variegata. The structures of these compounds were established by spectroscopic analysis. Eryvarin R (6) is an unusual 3-aryl-2,3-dihydrobenzofuran derivative with a formyl (CHO) group. Eryvarin Q (5) showed potent antibacterial activity against methicillin-resistant Staphylococcus aureus.     

The novel mutation K87E in ribosomal protein S12 enhances protein synthesis activity during the late growth phase in<Emphasis Type="Italic"> Escherichia coli</Emphasis>

Resistance to streptomycin in bacterial cells often results from a mutation in the rpsL gene that encodes the ribosomal protein S12. We found that a particular rpsL mutation (K87E), newly identified in Escherichia coli, causes aberrant protein synthesis activity late in the growth phase. While protein synthesis decreased with age in cells in the wild-type strain, it was sustained at a high level in the mutant, as determined using living cells. This was confirmed using an in vitro protein synthesis system with poly(U) and natural mRNAs (GFP mRNA and CAT mRNA). Other classical rpsL mutations (K42N and K42T) tested did not show such an effect, indicating that this novel characteristic is typical of ribosomes bearing the K87E mutant form of S12, although the K87E mutation conferred the streptomycin resistance and error-restrictive phenotypes also seen with the K42N and K42T mutations. The K87E (but not K42N or K42T) mutant ribosomes exhibited increased stability of the 70S complex in the presence of low concentrations of magnesium. We propose that the aberrant activation of protein synthesis at the late growth phase is caused by the increased stability of the ribosome.Communicated by W. Goebel     

An estrogen receptor (ER)alpha deoxyribonucleic acid-binding domain knock-in mutation provides evidence for nonclassical ER pathway signaling in vivo

We created a nonclassical estrogen receptor (ER) knock-in mouse model by introducing a mutation that selectively eliminates classical ER signaling through estrogen response elements, while preserving the nonclassical ER pathway. Heterozygous nonclassical ER knock-in (NERKI) females are infertile. Their ovaries contain no corpora lutea, reflecting a defect in ovulation, and the stromal cells contain lipid droplets, suggesting altered steroidogenesis. The uteri are enlarged with evidence of cystic endometrial hyperplasia, and the mammary glands are hypoplastic. These phenotypic features indicate differential ER effects on growth and development in various estrogen-responsive tissues. These findings suggest that nonclassical ER signaling pathways play an important physiological role in the development and function of the reproductive system.     

Possible antitumor promoters in Spinacia oleracea (spinach) and comparison of their contents among cultivars

Spinach leaves were found to contain two potent antitumor promoters as detected by the activity against tumor promoter-induced Epstein-Barr virus activation. The active components were identified as 1-O-alpha-linolenoyl-2-O-(7Z,10Z,13Z)-hexadecatrienoyl-3-O-beta-D-galactopyranosyl-sn-glycerol and 1,2-di-O-alpha-linolenoyl-3-O-beta-D-galactopyranosyl-sn-glycerol by spectroscopic data and some chemical and enzymatic reactions. Their contents significantly varied with the cultivar and with the culture conditions.     

Differentiation in the status of self-incompatibility among Calibrachoa species (Solanaceae)

The overall status of self-incompatibility, as assessed by the rate of capsule-set after self-pollination, was investigated in the genus Calibrachoa (Solanaceae). Thirty-two species were surveyed using a total of 655 individuals collected in 102 different native populations in Argentina, Brazil, Mexico, and Uruguay. The rate of capsule-set in 278 voucher specimens collected from the same native habitats was also measured to obtain additional information to assess the degree of self-(in)compatibility. Only one species, Calibrachoa parviflora, was self-compatible (SC, autogamous) and the other 31 species were found to be self-incompatible (SI). A mixed population (SI and SC individuals in the same population) was not found. The differentiation of C. parviflora as an autogamous species is associated with a successful occupation of different (riparian) habitats within a larger range of geographic distribution compared to the rest of the species in the principally SI genus of Calibrachoa.     

Effect of the V3 loop deletion of envelope glycoprotein on cellular responses and protection against challenge with recombinant vaccinia virus expressing gp160 of primary human immunodeficiency virus type 1 isolates

The magnitude and breadth of cytotoxic-T-lymphocyte (CTL) responses induced by human immunodeficiency virus type 1 (HIV-1) envelope protein from which the hypervariable V3 loop had been deleted (DeltaV3) were evaluated in the HLA-A2/K(b) transgenic mice. It was demonstrated that vaccines expressing the DeltaV3 mutant of either HIV-1(IIIB) or HIV-1(89.6) envelope glycoprotein induced broader CD8(+) T-cell activities than those elicited by the wild-type (WT) counterparts. Specifically, the differences were associated with higher responses to conserved HLA-A2-restricted CTL epitopes of the envelope glycoprotein and could be correlated with an increased cell surface occupancy by the epitope-HLA-A2 complexes in target cells expressing the DeltaV3 mutant. Using recombinant vaccinia virus expressing heterologous gp160 of primary HIV-1 isolates in a murine challenge system, we observed that the extent of resistance to viral transmission was higher in animals immunized with the DeltaV3 than the WT envelope vaccine. The protection was linked to the presence of envelope-specific CD8(+) T cells, since depletion of these cells by anti-CD8 antibody treatment at the time of challenge abolished the vaccine-induced protection. The results from our studies provide insights into approaches for boosting the breadth of envelope-specific CTL responses.     

Pretreatment procedure for the microdetermination of chondroitin sulfate in plasma and urine

A new, simple, and rapid pretreatment method for the determination of chondroitin sulfate, dermatan sulfate, and hyaluronan from urine and blood plasma samples has been developed. Plasma proteins were first converted into small peptides by digestion using a nonspecific protease, actinase E, and the resulting small peptides were removed by centrifugal filtration. The retained, residual crude glycosaminoglycans, including chondroitin/dermatan sulfates and hyaluronan, were converted into unsaturated disaccharides through the action of chondroitin sulfate lyses. Next, these disaccharides were recovered and purified using centrifugal filtration together with DeltaDi-UA2S, added as an internal standard. The filtered disaccharide mixture was analyzed by HPLC with fluorometric postcolumn derivatization using 2-cyanoacetamide as a fluorogenic reagent. This method was applied to a pharmacokinetic study of chondroitin sulfate administered intravenously to mice. The half-life of the administered chondroitin sulfates, having molecular masses from 6 to 50 kDa, varied depending on their molecular sizes. This new method should be useful for studies on the metabolic fate of exogenously administered glycosaminoglycans in small experimental animals.     

Analysis of human TIE2 function on hematopoietic stem cells in umbilical cord blood

To investigate the behavior of hematopoietic stem cells (HSCs) in cord blood (CB), we analyzed the expression and function of TIE2, a tyrosine kinase receptor. A subpopulation of Lineage (Lin)(-/low)CD34(+) cells in CB expressed TIE2 (18.8%). Assays for long-term culture-initiating cells (LTC-IC) and cobble-stone formation revealed that Lin(-/low)CD34(+)TIE2(+) cells showed to have a capacity of primitive hematopoietic precursor cells in vitro. When Lin(-/low)CD34(+)TIE2(+) cells were cultured on the stromal cells, they transmigrated under the stromal layers and kept an immature character for a few weeks. By contrast, Lin(-/low)CD34(+)TIE2(-) cells differentiated immediately within a few weeks. Finally, we confirmed that 1x10(4)Lin(-/low)CD34(+)TIE2(+) cells were engrafted in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, while 1x10(4)Lin(-/low)CD34(+)TIE2(-) cells were not. Taken together, we conclude that TIE2 is a marker of HSCs in CB. A ligand for TIE2, Ang-1 promoted the adhesion of sorted primary Lin(-/low)CD34(+)TIE2(+) cells to fibronectin (FN), and this adhesion may play a critical role in keeping HSCs in an immature status under the stromal cells.