Macrophage chemotactic factor (MCF) produced by a human T cell hybridoma clone

A human T cell hybridoma clone, D6-18, producing high levels of macrophage chemotactic factor (MCF) was established by the emetine-actinomycin D selection method. MCF was found to be present not only in the culture medium but also in the cell lysate of D6-18 cells. The secretion of the MCF from D6-18 cells was effectively inhibited by disodium cromoglycate, which is an inhibitor of the degranulation of mast cells, suggesting that MCF is stored in granules. The MCF of D6-18 cells was purified from the sonicated cell lysate by ion-exchange chromatographies and high-performance liquid chromatography. The amino acid sequence of the purified MCF was revealed to be WLGREDGSE or WLGRQDGSE. The synthetic peptide WLGREDGSE showed chemotactic activity against guinea pig macrophages and human monocytes at the concentration of about 10(-8) M.     

Endothelial specific granules in the umbilical veins of the postnatal rabbit

Summary A remarkable increase in number of endothelial specific granules was observed in the rabbit umbilical veins between 2 and 5 days after birth. Electron microscopy indicated that the granules were segregated in the Golgi complex of the endothelial cells and released into the vascular lumen during the postnatal obliteration stage of this vessel.Incubation of the postnatal vessels in Ringer solution containing a histamine releasing compound induced remarkable morphological alterations of these cytoplasmic components; a reduction of their osmiophilia, swelling with a widened space separating the granular matrix from the limiting membrane, fusion to each other and expulsion of their contents into the vascular lumen, as in mast cell degranulation by this drug, were noted.High-performance liquid chromatography of the homogenized vessels demonstrated appreciable concentrations of histamine in the postnatal samples. There was a correlation between the histamine concentration and the quantity of granules in the respective postnatal samples.The present study strongly suggests that the granules are reservoirs of histamine and have an important role in the obliteration of this vessel.This work was supported in part by Grant in Aid for Scientific Research (# 448087) to S. Fujimoto from the Ministry of Education of Japan     

Terpenoids from Tripterygium doianum (Celastraceae)

The extract of Tripterygium doianum (Celastraceae) afforded three triterpenoids [3beta-acetoxy-11-ursen-13alpha,30-olide, 25-chloro-24-hydroxytirucall-7-en-3-one and tirucall-7-en-3,24-dione], two sesquiterpenoids [5alpha-acetoxy-1beta,8alpha-bis-cinnamoyl-4alpha-hydroxydihydroagarofuran and 5alpha-acetoxy-1beta-benzoyl-8alpha-cinnamoyl-4alpha-hydroxydihydroagarofuran] and nine known triterpenoids. Their structures were established based on spectroscopic studies.     

Interannual variations in abundance and body size in Neocalanus copepods in the central North Pacific

As the integral components of zooplankton in the subarctic NorthPacific, the three Neocalanus species (N. cristatus, N. plumchrusand N. flemingeri) are characterized by an annual life cycleand rapid development in the surface layer during spring–summer.Patterns of interannual variation of abundance and body sizeof these Neocalanus species were analyzed using the time-seriesdata collected during the summers of 1979–1998 (20 years)at stations along the longitudinal transect line in the centralNorth Pacific, crossing five sub-areas (Alaska Current System,Subarctic Current System, Northern Transition Domain, SouthernTransition Domain and Subtropical Current System). In the southernsub-areas, quasi-decadal oscillation was observed for the 3-yearrunning mean of abundance and prosome length for copepoditestage 5 (C5) of the three Neocalanus species. Although the oscillationsignal diminished towards northern waters, it showed a positivephase during the early 1980s and 1990s and a negative phaseduring the late 1980s. In the northern waters, a biennial patternwas pronounced for anomalies of C5 prosome length for N. plumchrusand N. flemingeri, which was large in odd years and small ineven years. Significantly positive covariations among the threespecies were found for both abundance and prosome length aroundmid-latitude, where they were abundant. In the correlation analysis,these observed yearly patterns showed a statistically insignificantcorrelation with most environmental (integrated mean temperaturein surface waters, water column stability and chlorophyll aconcentration) or climatological (North Pacific Index and SouthernOscillation Index) variables. The regional difference of theoscillation signal and the synchronized covariation among thesespecies suggest that interannual variations of their abundanceand body size are mediated by common environmental force(s)with some spatial and temporal scales in the subarctic NorthPacific.     

Structural basis for acceptor‐substrate recognition of UDP‐glucose: anthocyanidin 3‐O‐glucosyltransferase from Clitoria ternatea

UDP‐glucose: anthocyanidin 3‐O‐glucosyltransferase (UGT78K6) from Clitoria ternatea catalyzes the transfer of glucose from UDP‐glucose to anthocyanidins such as delphinidin. After the acylation of the 3‐O‐glucosyl residue, the 3′‐ and 5′‐hydroxyl groups of the product are further glucosylated by a glucosyltransferase in the biosynthesis of ternatins, which are anthocyanin pigments. To understand the acceptor‐recognition scheme of UGT78K6, the crystal structure of UGT78K6 and its complex forms with anthocyanidin delphinidin and petunidin, and flavonol kaempferol were determined to resolutions of 1.85 Å, 2.55 Å, 2.70 Å, and 1.75 Å, respectively. The enzyme recognition of unstable anthocyanidin aglycones was initially observed in this structural determination. The anthocyanidin‐ and flavonol‐acceptor binding details are almost identical in each complex structure, although the glucosylation activities against each acceptor were significantly different. The 3‐hydroxyl groups of the acceptor substrates were located at hydrogen‐bonding distances to the Nε2 atom of the His17 catalytic residue, supporting a role for glucosyl transfer to the 3‐hydroxyl groups of anthocyanidins and flavonols. However, the molecular orientations of these three acceptors are different from those of the known flavonoid glycosyltransferases, VvGT1 and UGT78G1. The acceptor substrates in UGT78K6 are reversely bound to its binding site by a 180° rotation about the O1–O3 axis of the flavonoid backbones observed in VvGT1 and UGT78G1; consequently, the 5‐ and 7‐hydroxyl groups are protected from glucosylation. These substrate recognition schemes are useful to understand the unique reaction mechanism of UGT78K6 for the ternatin biosynthesis, and suggest the potential for controlled synthesis of natural pigments.     

An alternative transcript derived from the trio locus encodes a guanosine nucleotide exchange factor with mouse cell-transforming potential

By screening cDNA expression libraries derived from fresh leukemic cells of adult T-cell leukemia for the potential to transform murine fibroblasts, NIH3T3, we have identified a novel transforming gene, designated Tgat. Expression of Tgat in NIH3T3 resulted in the loss of contact inhibition, increase of saturation density, anchorage-independent growth in a semisolid medium, tumorigenicity in nude mice, and increased invasiveness. Sequence comparison revealed that an alternative RNA splicing of the Trio gene was involved in the generation of Tgat. The Tgat cDNA encoded a protein product consisting of the Rho-guanosine nucleotide exchange factor (GEF) domain of a multifunctional protein, TRIO, and a unique C-terminal 15-amino acid sequence, which were derived from the exons 38-46 of the Trio gene and a novel exon located downstream of its last exon (exon 58), respectively. A Tgat mutant cDNA lacking the C-terminal coding region preserved Rho-GEF activity but lost the transforming potential, indicating an indispensable role of the unique sequence. On the other hand, treatment of Tgat-transformed NIH3T3 cells with Y-27632, a pharmacological inhibitor of Rho-associated kinase, abrogated their transforming phenotypes, suggesting the coinvolvement of Rho-GEF activity. Thus, alternative RNA splicing, resulting in the fusion protein with the Rho-GEF domain and the unique 15 amino acids, is the mechanism generating the novel oncogene, Tgat.     

Effects of fifteen rare-earth metals on Ca2+ influx in tobacco cells

Effects of naturally existing rare-earth metals (REMs; atomic numbers, 39, 57-60, 62-71; Y, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu), added as chloride salts, on Ca2+ influx induced by two different stimuli, namely hypoosmotic shock and hydrogen peroxide, were examined in a suspension-cultured transgenic cell line of BY-2 tobacco cells expressing aequorin, a Ca(2+)-sensitive luminescent protein in cytosol. Most REM salts used here showed inhibitory effect against Ca2+ influx. Especially NdCl3, SmCl3, EuCl3, GdCl3 and TbCl3 showed the most robust inhibitory action. In contrast, LuCl3, YbCl3, ErCl3 and YCl3 were shown to be poor inhibitors of Ca2+ influx. Since REMs tested here form a sequential range of ionic radii from 86.1 to 103.2 pm and the optimal range of ionic radii required for blocking the flux of Ca2+ was determined for each stimulus. The hydrogen peroxide-induced Ca2+ influx was optimally blocked by REMs with a broad range of ionic radii (93.8-101 pm) which is slightly smaller than or similar to that of Ca2+ (100 pm), while the hypoosmotically induced flux of Ca2+ was inhibited optimally by few REMs with a narrower range of relatively smaller ionic radii around that of Gd3+ (93.8 pm) a well known inhibitor of stretch-activated channels. Possible applications of such series of channel blockers in elucidation of plant signal transduction pathways are encouraged.     

Impaired autophagy by soluble endoglin,under physiological hypoxia in early pregnant period,is involved in poor placentation in preeclampsia

In early pregnancy, trophoblasts and the fetus experience hypoxic and low-nutrient conditions; nevertheless, trophoblasts invade the uterine myometrium up to one third of its depth and migrate along the lumina of spiral arterioles, replacing the maternal endothelial lining. Here, we showed that autophagy, an intracellular bulk degradation system, occurred in extravillous trophoblast (EVT) cells under hypoxia in vitro and in vivo. An enhancement of autophagy was observed in EVTs in early placental tissues, which suffer from physiological hypoxia. The invasion and vascular remodeling under hypoxia were significantly reduced in autophagy-deficient EVT cells compared with wild-type EVT cells. Interestingly, soluble endoglin (sENG), which increased in sera in preeclamptic cases, suppressed EVT invasion by inhibiting autophagy. The sENG-inhibited EVT invasion was recovered by TGFB1 treatment in a dose-dependent manner. A high dose of sENG inhibited the vascular construction by EVT cells and human umbilical vein endothelial cells (HUVECs), meanwhile a low dose of sENG inhibited the replacement of HUVECs by EVT cells. A protein selectively degraded by autophagy, SQSTM1, accumulated in EVT cells in preeclamptic placental biopsy samples showing impaired autophagy. This is the first report showing that impaired autophagy in EVT contributes to the pathophysiology of preeclampsia.     

An Atg4B mutant hampers the lipidation of LC3 paralogues and causes defects in autophagosome closure

In the process of autophagy, a ubiquitin-like molecule, LC3/Atg8, is conjugated to phosphatidylethanolamine (PE) and associates with forming autophagosomes. In mammalian cells, the existence of multiple Atg8 homologues (referred to as LC3 paralogues) has hampered genetic analysis of the lipidation of LC3 paralogues. Here, we show that overexpression of an inactive mutant of Atg4B, a protease that processes pro-LC3 paralogues, inhibits autophagic degradation and lipidation of LC3 paralogues. Inhibition was caused by sequestration of free LC3 paralogues in stable complexes with the Atg4B mutant. In mutant overexpressing cells, Atg5- and ULK1-positive intermediate autophagic structures accumulated. The length of these membrane structures was comparable to that in control cells; however, a significant number were not closed. These results show that the lipidation of LC3 paralogues is involved in the completion of autophagosome formation in mammalian cells. This study also provides a powerful tool for a wide variety of studies of autophagy in the future.