Establishment and characterization of a new human cell line (EJ) derived from endometrial carcinoma

We present a new cell line, EJ established from an invasive endometrioid adenocarcinoma of the uterine corpus in a 56-year-old patient. The cells show rapid growth in culture with a doubling time of 16 h and high migration activity. Monolayer-cultured cells were polygonal in shape showing a tendency to pile up without contact inhibition. Subcutaneous transplantation of the EJ cells into nude mice formed solid tumors that were histologically diagnosed as adenocarcinoma, whereas no metastasis was observed. Cultured EJ cells produced tissue polypeptide antigen (IPA). Genetic and molecular analyses revealed high telomerase activity but not estrogen receptor alpha expression. Using the DNA sequencing technique, we have screened EJ cells for p53 mutation in exon 5 to 8 but no mutation of p53 was observed. This cell line appears to represent the development of a more malignant clone with divergent receptor function and growth behavior, and provides us with an interesting new tool for the study of tumorigenesis in the human endometrium.     

Distinctive response of thyroid-infiltrating mononuclear cells to B cell activation through CD40 and interleukin-4 in Graves' patients

The possible role of abnormal T cell-dependent B-cell activation in Graves' disease was investigated by comparing lymphocyte subset distribution and the production of soluble CD8 (sCD8), sCD23, IL-10 and IL-12 by peripheral blood cells (PBMC) and thyroid-infiltrating lymphocytes (TL) in vitro. In TL, the percentage of CD8(+) cells was slightly higher and the sCD8 concentration was significantly higher than in PBMC. The ratio CD23(+) cells to CD20(+) cells (activated B/pan B cells) was increased in TL compared to PBMC from Graves' or normal controls, although the percentage of CD20(+) cells was decreased. Compared to PBMC in Graves' disease, the relative ratio of IL-10 to IL-12 release (IL-10/IL-12) by unstimulated TL was increased, despite a lack of significant difference between PBMC and TL in mean values for either IL-10 or IL-12 secretion. Incubating PBMC with a combination of anti-CD40 monoclonal antibodies and interleukin-4 (IL-4) resulted in B cell activation, reflected in an increase in the sCD23 level in both controls and Graves' patients, but especially prominent in the latter. Stimulation with anti-CD40 antibody and IL-4 also decreased the percentage of CD8(+) cells in PBMC but not TL from both Graves' disease and normal controls, and the percentage of CD8(+) cells in TL was higher than PBMC after the stimulation. The sCD23 concentration in TL was decreased compared to PBMC both in patients with Graves' disease and normal controls. However, in contrast to the increased responses observed in Graves' PBMC or normal controls after stimulation, sCD23 levels remained the same in stimulated TL from Graves' patients. This combination of B cell stimulants increased production of IL-10 in PBMC but not in TL obtained from patients with Graves' disease, and the increased IL-10/IL-12 ratio declined to a value no different from that in PBMC group after stimulation. Thus, T cell-dependent B-cell activation via a CD40 pathway may cause a shift in the Th(1)/Th(2) balance to Th(2) dominance in Graves' disease, while increased CD8(+) cells in TL may suppress sCD23 production and IL-10-producing Th(2) cells.     

Detection of non-Brownian diffusion in the cell membrane in single molecule tracking

Molecules undergo non-Brownian diffusion in the plasma membrane, but the mechanism behind this anomalous diffusion is controversial. To characterize the anomalous diffusion in the complex system of the plasma membrane and to understand its underlying mechanism, single-molecule/particle methods that allow researchers to avoid ensemble averaging have turned out to be highly effective. However, the intrinsic problems of time-averaging (resolution) and the frequency of the observations have not been explored. These would not matter for the observations of simple Brownian particles, but they do strongly affect the observation of molecules undergoing anomalous diffusion. We examined these effects on the apparent motion of molecules undergoing simple, totally confined, or hop diffusion, using Monte Carlo simulations of particles undergoing short-term confined diffusion within a compartment and long-term hop diffusion between these compartments, explicitly including the effects of time-averaging during a single frame of the camera (exposure time) and the frequency of observations (frame rate). The intricate relationships of these time-related experimental parameters with the intrinsic diffusion parameters have been clarified, which indicated that by systematically varying the frame time and rate, the anomalous diffusion can be clearly detected and characterized. Based on these results, single-particle tracking of transferrin receptor in the plasma membrane of live PtK2 cells were carried out, varying the frame time between 0.025 and 33 ms (0.03-40 kHz), which revealed the hop diffusion of the receptor between 47-nm (average) compartments with an average residency time of 1.7 ms, with the aid of single fluorescent-molecule video imaging.     

Synergy and antagonism between Notch and BMP receptor signaling pathways in endothelial cells

Notch and bone morphogenetic protein signaling pathways are important for cellular differentiation, and both have been implicated in vascular development. In many cases the two pathways act similarly, but antagonistic effects have also been reported. The underlying mechanisms and whether this is caused by an interplay between Notch and BMP signaling is unknown. Here we report that expression of the Notch target gene, Herp2, is synergistically induced upon activation of Notch and BMP receptor signaling pathways in endothelial cells. The synergy is mediated via RBP-Jkappa/CBF-1 and GC-rich palindromic sites in the Herp2 promoter, as well as via interactions between the Notch intracellular domain and Smad that are stabilized by p/CAF. Activated Notch and its downstream effector Herp2 were found to inhibit endothelial cell (EC) migration. In contrast, BMP via upregulation of Id1 expression has been reported to promote EC migration. Interestingly, Herp2 was found to antagonize BMP receptor/Id1-induced migration by inhibiting Id1 expression. Our results support the notion that Herp2 functions as a critical switch downstream of Notch and BMP receptor signaling pathways in ECs.     

Identification and characterization of cDNAs encoding pentatricopeptide repeat proteins in the basal land plant, the moss Physcomitrella patens

A large gene family encoding proteins with a pentatricopeptide repeat (PPR) motif exists in flowering plants but not in algae, fungi, or animals. This suggests that PPR protein genes expanded vastly during the evolution of the land plants. To investigate this possibility, we analysed PPR protein genes in the basal land plant, the moss Physcomitrella patens. An extensive survey of the Physcomitrella expressed sequence tag (EST) databases revealed 36 ESTs encoding PPR proteins. This indicates that a large gene family of PPR proteins originated before the divergence of the vascular plant and moss lineages. We also characterized five full-length cDNAs encoding PPR proteins, designated PPR513-10, PPR566-6, PPR868-14, PPR986-12, and PPR423-6. Intracellular localization analysis demonstrated two PPR proteins in chloroplasts (cp), whereas the cellular localization of the other three PPR proteins is unclear. The genes of the cp-localized PPR513-10 and PPR566-6 were expressed differentially in protonemata grown under different light-dark conditions, suggesting they have distinctive functions in cp. This is the first report and analysis of genes encoding PPR proteins in bryophytes.     

Possible involvement of p38 MAP kinase in prostaglandin E1-induced ALP activity in osteoblast-like cells

Prostaglandins are now recognized to be important regulators for both bone formation and resorption. Among them, prostaglandin E(1) (PGE(1)) has been reported to stimulate cAMP accumulation and to induce alkaline phosphatase (ALP) activity, a marker of differentiation, in osteoblast-like cells. Recently, we have shown that p38 mitogen-activated protein (MAP) kinase pathway regulates ALP activity in response to activation of Gi protein-coupled receptors in mouse osteoblast-like MC3T3-E1 cells (Suzuki et al., Endocrinology 140 (1999) 3177). In the present study, we investigated whether p38 MAP kinase is involved in ALP activation by PGE(1) in MC3T3-E1 osteoblast-like cells. PGE(1) dose-dependently enhanced ALP activities in the concentration range between 1 nM and 1 microM in MC3T3-E1 cells. SB203580, a specific inhibitor of p38 MAP kinase, blocked the increase in ALP activity induced by PGE(1). Further analysis with western blotting suggested that PGE(1) induced an increase in tyrosine (Tyr) phosphorylation of p38 MAP kinase. Both Bt(2)cAMP, a permeable analogue of cAMP, and forskolin, which directly activates adenylate cyclase, also induced an increase in Tyr phosphorylation of p38 MAP kinase. H-89, a potent inhibitor of protein kinase A (PKA), significantly suppressed PGE(1)-induced Tyr phosphorylation of p38 MAP kinase. The results of this study suggest that PGE(1) stimulates p38 MAP kinase through the activation of PKA, resulting in the enhancement of ALP activity.     

Physcomitrella PpORS,Basal to Plant Type III Polyketide Synthases in Phylogenetic Trees,Is a Very Long Chain 2′-Oxoalkylresorcinol Synthase

The plant type III polyketide synthases (PKSs), which produce diverse secondary metabolites with different biological activities, have successfully co-evolved with land plants. To gain insight into the roles that ancestral type III PKSs played during the early evolution of land plants, we cloned and characterized PpORS from the moss Physcomitrella. PpORS has been proposed to closely resemble the most recent common ancestor of the plant type III PKSs. PpORS condenses a very long chain fatty acyl-CoA with four molecules of malonyl-CoA and catalyzes decarboxylative aldol cyclization to yield the pentaketide 2′-oxoalkylresorcinol. Therefore, PpORS is a 2′-oxoalkylresorcinol synthase. Structure modeling and sequence alignments identified a unique set of amino acid residues (Gln²¹⁸, Val²⁷⁷, and Ala²⁸⁶) at the putative PpORS active site. Substitution of the Ala²⁸⁶ to Phe apparently constricted the active site cavity, and the A286F mutant instead produced triketide alkylpyrones from fatty acyl-CoA substrates with shorter chain lengths. Phylogenetic analysis and comparison of the active sites of PpORS and alkylresorcinol synthases from sorghum and rice suggested that the gramineous enzymes evolved independently from PpORS to have similar functions but with distinct active site architecture. Microarray analysis revealed that PpORS is exclusively expressed in nonprotonemal moss cells. The in planta function of PpORS, therefore, is probably related to a nonprotonemal structure, such as the cuticle.     

Clinical and genetic studies of Japanese homozygotes for the Gaucher disease L444P mutation

In patients originally genotyped as homoallelic for the Gaucher disease (GD) L444P (1448C) mutation, we sought to confirm previously reported phenotypic differences between Caucasians and Japanese, to determine the prevalence and phenotypic impact of recombinant alleles, and to explore the phenotypic influence of genetic background. We therefore analyzed data from longer-term clinical follow-up, more comprehensive genotyping and polymorphism and mitochondrial DNA (mtDNA) testing in all known Japanese L444P homozygotes (n=15). Our studies demonstrated that, of 12 patients in our series originally diagnosed with non-neuronopathic GD, 9 developed neurological signs/symptoms during follow-up (at a mean of 14 years 11 months±11 years 4 months). Of three patients originally diagnosed with acute neuronopathic (type 2) GD, all three were compound heterozygotes for L444P and the complex allele RecNci I. In the entire series, Pvu II and liver erythrocyte pyruvate kinase (PKLR) polymorphism and prevalence of the 9 bp mtDNA deletion were heterogeneous, and these background genetic factors could not predict phenotypic expression. Our data suggest that, in Japanese as in Caucasian patients, the L444P/L444P genotype is highly associated with subacute neuronopathic (type 3) GD, and the presence of a complex allele together with an L444P allele leads to type 2 disease. Our findings also underline the importance of comprehensive genotyping (particularly testing for recombinant alleles), long-term follow-up and careful neurological examination in patients with early-onset GD. Such measures ultimately may improve genotype/phenotype correlations and, with them, genetic counseling and therapeutic decision making. Electronic Publication