First determination of the inhibitor complex structure of human hematopoietic prostaglandin D synthase

Hematopoietic prostaglandin (PG) D synthase (H-PGDS) is responsible for the production of PGD(2) as an allergy or inflammation mediator in mast and Th2 cells. We determined the X-ray structure of human H-PGDS complexed with an inhibitor, 2-(2'-benzothiazolyl)-5-styryl-3-(4'-phthalhydrazidyl) tetrazolium chloride (BSPT) at 1.9 A resolution in the presence of Mg(2+). The styryl group of the inhibitor penetrated to the bottom of the active site cleft, and the tetrazole ring was stabilized by the stacking interaction with Trp104, inducing large movement around the alpha5-helix, which caused the space group of the complex crystal to change from P2(1) to P1 upon binding of BSPT. The phthalhydrazidyl group of BSPT exhibited steric hindrance due to the cofactor, glutathione (GSH), increasing the IC(50) value of BSPT for human H-PGDS from 36.2 micro M to 98.1 micro M upon binding of Mg(2+), because the K(m) value of GSH for human H-PGDS was decreased from 0.60 micro M in the presence of EDTA to 0.14 micro M in the presence of Mg(2+). We have to avoid steric hindrance of the GSH molecule that was stabilized by intracellular Mg(2+) in the mM range in the cytosol for further development of structure-based anti-allergic drugs.     

FRL-1, a member of the EGF-CFC family,is essential for neural differentiation in Xenopus early development

Recent studies indicate an essential role for the EGF-CFC family in vertebrate development, particularly in the regulation of nodal signaling. Biochemical evidence suggests that EGF-CFC genes can also activate certain cellular responses independently of nodal signaling. Here, we show that FRL-1, a Xenopus EGF-CFC gene, suppresses BMP signaling to regulate an early step in neural induction. Overexpression of FRL-1 in animal caps induced the early neural markers zic3, soxD and Xngnr-1, but not the pan-mesodermal marker Xbra or the dorsal mesodermal marker chordin. Furthermore, overexpression of FRL-1 suppressed the expression of the BMP-responsive genes, Xvent-1 and Xmsx-1, which are expressed in animal caps and induced by overexpressed BMP-4. Conversely, loss of function analysis using morpholino-antisense oligonucleotides against FRL-1 (FRL-1MO) showed that FRL-1 is required for neural development. FRL-1MO-injected embryos lacked neural structures but contained mesodermal tissue. It was suggested previously that expression of early neural genes that mark the start of neuralization is activated in the presumptive neuroectoderm of gastrulae. FRL-1MO also inhibited the expression of these genes in dorsal ectoderm, but did not affect the expression of chordin, which acts as a neural inducer from dorsal mesoderm. FRL-1MO also inhibited the expression of neural markers that were induced by chordin in animal caps, suggesting that FRL-1 enables the response to neural inducing signals in ectoderm. Furthermore, we showed that the activation of mitogen-activated protein kinase by FRL-1 is required for neural induction and BMP inhibition. Together, these results suggest that FRL-1 is essential in the establishment of the neural induction response.     

Xenopus tropicalis nodal-related gene 3 regulates BMP signaling: an essential role for the pro-region

In vertebrates, nodal-related genes are crucial for specifying mesendodermal cell fates. Six nodal-related genes have been identified in Xenopus, but only one, nodal, has been identified in the mouse. The Xenopus nodal-related gene 3 (Xnr3), however, lacks the mesoderm-inducing activity of the other five nodal-related genes in Xenopus, and can directly induce neural tissue in animal caps by antagonizing BMP signals. In this study, we isolated three clones of the Xenopus (Silurana) tropicalis nodal-related gene 3 (Xtnr3) and analyzed their function. The Xtnr3 genes show high homology to Xnr3 and have the same activity. Southern blot and genomic PCR analyses indicate that the X. tropicalis genome has duplications in the Xtnr3 gene sequences and our three clones represent separate gene loci. We also found a partial clone of Xtnr3 that coded for the N-terminal part of its pro-region. Surprisingly, this sequence also induced neural tissue by antagonizing BMP signals, and its coded protein physically associated with BMP4 mature protein. Furthermore, we showed that the pro-region of Xnr5 has the same activity. Together, these findings indicate that the pro-region of nodal-related genes acts antagonistically towards BMP signals, which identifies a novel mechanism for the inhibition of BMP signaling.     

A large variation in the rates of synonymous substitution for RNA viruses and its relationship to a diversity of viral infection and transmission modes

RNA viruses successfully adapt to various environments by repeatedly producing new mutants, often through generating a number of nucleotide substitutions. To estimate the degree of variation in mutation rates of RNA viruses and to understand the source of such variation, we studied the synonymous substitution rate because synonymous substitution is exempt from functional constraints at the protein level, and its rate reflects the mutation rate to a great extent. We estimated the synonymous substitution rates for a total of 49 different species of RNA viruses, and we found that the rates had tremendous variation by 5 orders of magnitude (from 1.3 x 10(-7) to 6.2 x 10(-2) /synonymous site/year). Comparing the synonymous substitution rates with the replication frequencies and replication error rates for the RNA viruses, we found that the main source of the rate variation was differences in the replication frequency because the rates of replication error were roughly constant over different RNA viruses. Moreover, we examined a relationship between viral life strategies and synonymous substitution rates to understand which viral life strategies affect replication frequencies. The results show that the variation of synonymous substitution rates has been influenced most by either the difference in the infection modes or the differences in the transmission modes. In conclusion, the variation of mutation rates for RNA viruses is caused by different replication frequencies, which are affected strongly by the infection and transmission modes.     

Phosphate Deficiency in Maize. IV. Changes in Amounts of Sucrose Phosphate Synthase during the Course of Phosphate Deprivation

With low-P treatment of maize, the level of sucrose phosphatesynthase (SPS) protein decreased to 15% of the control, whilethe "V_max" activity stayed relatively high (100–80% ofthe control) and the substrate limiting activity increased about2 fold in the leaves. These results suggest that leaf phosphatestatus has dual effects on the amount of SPS protein and theactivation state of SPS. (Received January 20, 1992; Accepted April 14, 1993)     

Novel distribution of adrenomedullin-immunoreactive cells in human tissues

Adrenomedullin (AM) is a novel hypotensive and vasodilator peptide. We previously examined the localization of AM in human, rat, and porcine tissues using a polyclonal antibody against synthetic human AM[40–52]. We demonstrated that AM is widely distributed in the endocrine and neuroendocrine systems, but not in the heart, kidney, or blood vessels, although high levels of AM mRNA were detected in the latter tissues. In this study, we further investigated the distribution of AM by using two newly developed monoclonal antibodies against synthetic human AM peptides, [12–25] and [46–52]. AM immunoreactivity was observed in cardiac myocytes, vascular smooth muscle cells, endothelial cells, and renal distal and collecting tubules. In addition, AM-immunoreactive (IR) cells were found in mucosal and glandular epithelia of the digestive, respiratory, and reproductive systems, as well as the endocrine and neuroendocrine systems. These findings indicate that AM-IR cells are more widely distributed in human tissues and suggest that AM might play multiple biological roles in humans. Accepted: 7 June 1999     

Inhibitory Mechanism of FAT4 Gene Expression in Response to Actin Dynamics during Src-Induced Carcinogenesis

Oncogenic transformation is characterized by morphological changes resulting from alterations in actin dynamics and adhesive activities. Emerging evidence suggests that the protocadherin FAT4 acts as a tumor suppressor in humans, and reduced FAT4 gene expression has been reported in breast and lung cancers and melanoma. However, the mechanism controlling FAT4 gene expression is poorly understood. In this study, we show that transient activation of the Src oncoprotein represses FAT4 mRNA expression through actin depolymerization in the immortalized normal human mammary epithelial cell line MCF-10A. Src activation causes actin depolymerization via the MEK/Erk/Cofilin cascade. The MEK inhibitor U0126 blocks the inhibitory effect of Src on FAT4 mRNA expression and Src-induced actin depolymerization. To determine whether actin dynamics act on the regulation of FAT4 mRNA expression, we treated MCF-10A cells with the ROCK inhibitor Y-27632. Y-27632 treatment decreased FAT4 mRNA expression. This suppressive effect was blocked by siRNA-mediated knockdown of Cofilin1. Furthermore, simultaneous administration of Latrunculin A (an actin depolymerizing agent), Y-27632, and Cofilin1 siRNA to the cells resulted in a marked reduction of FAT4 mRNA expression. Intriguingly, we also found that FAT4 mRNA expression was reduced under both low cell density and low stiffness conditions, which suggests that mechanotransduction affects FAT4 mRNA expression. Additionally, we show that siRNA-mediated FAT4 knockdown induced the activity of the Hippo effector YAP/TAZ in MCF-10A cells. Taken together, our results reveal a novel inhibitory mechanism of FAT4 gene expression through actin depolymerization during Src-induced carcinogenesis in human breast cells.     

Deficiency of Calcium-Independent Phospholipase A2 Beta Induces Brain Iron Accumulation through Upregulation of Divalent Metal Transporter 1

Mutations in PLA2G6 have been proposed to be the cause of neurodegeneration with brain iron accumulation type 2. The present study aimed to clarify the mechanism underlying brain iron accumulation during the deficiency of calcium-independent phospholipase A2 beta (iPLA₂β), which is encoded by the PLA2G6 gene. Perl’s staining with diaminobenzidine enhancement was used to visualize brain iron accumulation. Western blotting was used to investigate the expression of molecules involved in iron homeostasis, including divalent metal transporter 1 (DMT1) and iron regulatory proteins (IRP1 and 2), in the brains of iPLA₂β-knockout (KO) mice as well as in PLA2G6-knockdown (KD) SH-SY5Y human neuroblastoma cells. Furthermore, mitochondrial functions such as ATP production were examined. We have discovered for the first time that marked iron deposition was observed in the brains of iPLA₂β-KO mice since the early clinical stages. DMT1 and IRP2 were markedly upregulated in all examined brain regions of aged iPLA₂β-KO mice compared to age-matched wild-type control mice. Moreover, peroxidized lipids were increased in the brains of iPLA₂β-KO mice. DMT1 and IRPs were significantly upregulated in PLA2G6-KD cells compared with cells treated with negative control siRNA. Degeneration of the mitochondrial inner membrane and decrease of ATP production were observed in PLA2G6-KD cells. These results suggest that the genetic ablation of iPLA₂β increased iron uptake in the brain through the activation of IRP2 and upregulation of DMT1, which may be associated with mitochondrial dysfunction.     

Sex-specific HDL cholesterol changes with weight loss and their association with anthropometric variables: the LIFE study

Decrease in the level of high-density lipoprotein cholesterol (HDLC) has been observed in women who start dieting, but not in men. Patterns of HDLC change during intentional weight loss through 30-months of follow-up, and their association with changes in anthropometric measurements were examined in obese women (N = 112) and men (N = 100). Missing HDLC values at 6-, 12-, 18-, and 30-month follow-up (N = 16, 34, 55, and 50, respectively) due to dropout were imputed by multiple imputation. Mean ages and BMIs of subjects at baseline were 47.2 years and 34.8 kg/m(2) for women, and 50.4 years and 35.0 kg/m(2) for men. On average, participants lost weight steadily for 12 months, followed by slow regain. During the first 6 months, HDLC decreased significantly in women (-4.1 mg/dl, P = 0.0007), but not in men. Significant HDLC increases were observed in both men and women from 6- to 12-month follow-up. HDLC changes in women were positively associated with changes in hip circumference from baseline to 12-month independent of changes in triglycerides (TG), glucose, and insulin. Rapid decrease of predominantly subcutaneous fat in the femoral and gluteal area might be associated with HDLC decrease in women during initial weight loss.