Synergistic activation of a family of phosphoinositide 3-kinase via G-protein coupled and tyrosine kinase-related receptors.

Phosphoinositide 3-kinase (PI 3-kinase) is a key signaling enzyme implicated in a variety of receptor-stimulated cell responses. Stimulation of receptors possessing (or coupling to) protein-tyrosine kinase activates heterodimeric PI 3-kinases, which consist of an 85-kDa regulatory subunit (p85) containing Src-homology 2 (SH2) domains and a 110-kDa catalytic subunit (p110 alpha or p110 beta). Thus, this form of PI 3-kinases could be activated in vitro by a phosphotyrosyl peptide containing a YMXM motif that binds to the SH2 domains of p85. Receptors coupling to alpha beta gamma-trimeric G proteins also stimulate the lipid kinase activity of a novel p110 gamma isoform, which is not associated with p85, and thereby is not activated by tyrosine kinase receptors. The activation of p110 gamma PI 3-kinase appears to be mediated through the beta gamma subunits of the G protein (G beta gamma). In addition, rat liver heterodimeric PI 3-kinases containing the p110 beta catalytic subunit are synergistically activated by the phosphotyrosyl peptide plus G beta gamma. Such enzymatic properties were also observed with a recombinant p110 beta/p85 alpha expressed in COS-7 cells. In contrast, another heterodimeric PI 3-kinase consisting of p110 alpha and p85 in the same rat liver, together with a recombinant p110 alpha/p85 alpha, was not activated by G beta gamma, though their activities were stimulated by the phosphotyrosyl peptide. Synergistic activation of PI 3-kinase by the stimulation of the two major receptor types was indeed observed in intact cells, such as chemotactic peptide (N-formyl-Met-Leu-Phe) plus insulin (or Fc gamma II) receptors in differentiated THP-1 and CHO cells and adenosine (A1) plus insulin receptors in rat adipocytes. Thus, PI 3-kinase isoforms consisting of p110 beta catalytic and SH2-containing (p85 or its related) regulatory subunits appeared to function as a 'cross-talk' enzyme between the two signal transduction pathways mediated through tyrosine kinase and G protein-coupled receptors.     

1,3-Disubstituted benzazepines as neuropeptide Y Y1 receptor antagonists.

A novel class of potent and selective non-peptide neuropeptide Y (NPY) Y1 receptor antagonists, having benzazepine nuclei, have been designed, synthesized, and evaluated for activity. Through a blind screening we found the compound 1-N-(3-(N'-(tert-butoxycarbonyl)amino)benzyl)-7-methoxy-(3-(3)-methyl ureido)-2,3,4,5-tetrahydro-1H-1-benzazepin-2-one (9: IC50 = 1.6 microM). Chemical modifications of 9 gave a potent NPY Y1 antagonist 3-(N-(4-hydroxyphenyl)-N'-methylguanidino)-1-N-(3-(N'-(tert-butoxy carbonyl)amino)benzyl)-2,3,4,5-tetrahydro-1H-1-benzazepin-2-one (14c: IC5(0=43 nM), which had no affinity for NPY Y2 and Y5 receptors.     

Estrogen effects on platelet-activating factor and platelet-activating factor acetylhydrolase activity in rat uterus during the late stages of pregnancy.

The platelet-activating factor (PAF) concentration of the uterus spontaneously increased during pregnancy. When 17alpha-ethynylestradiol (0.25 mg/kg) was administered subcutaneously to pregnant rats for 3 days starting on Day 17 of pregnancy, some rats delivered prematurely on Day 20. However, none of the vehicle-treated (80% dimethylsulfoxide and 20% ethanol) pregnant rats delivered prematurely. The PAF concentration of the uterus in pregnant rats treated with 17alpha-ethynylestradiol was significantly higher than in those treated with vehicle on Days 19 and 20. On the other hand, the specific activity of uterine PAF-acetylhydrolase (PAF-AH) in pregnant rats treated with 17alpha-ethynylestradiol was significantly lower than in those treated with vehicle on Days 19 and 20, and the plasma PAF-AH activity in pregnant rats treated with estrogen was also significantly lower than in treated with vehicle on Days 18, 19, and 20. These findings indicate that estrogen increases PAF concentrations in the rat uterus, and this was correlated with a decrease in PAF-AH in the uterus and plasma. The increase in PAF concentrations in the uterus may be related to premature delivery and labor caused by PAF's known effect on myometrial contraction.     

Thiazolidinedione inhibits production of RANTES in a cytokine-treated human lung epithelial cell line.

The chemokine RANTES is a potent chemoattractant for eosinophils. RANTES is produced by lung epithelial cells during eosinophil-rich inflammatory diseases such as asthma. In this study, we examined the effects of thiazolidinediones (TZD) on RANTES expression in a human lung epithelial cell line, A549. In A549 cells, interleukin-1beta and tumor necrosis factor-alpha induced endogenous RANTES protein secretion, mRNA expression, and promoter activity. The TZD inhibited these effects. Our data indicate that the suppression of the expression of RANTES can be accomplished by TZD treatment, raising the possibility that TZD might be of therapeutic value in diseases such as asthma.     

Insulin-induced desensitization of extracellular signal-regulated kinase activation results from an inhibition of Raf activity independent of Ras activation and dissociation of the Grb2-SOS complex.

Previous studies have suggested that the interaction between the small adaptor protein Grb2 with the Ras guanyl nucleotide exchange factor SOS is functionally important in the regulation of the Ras activation/inactivation cycle. To examine the relationship between the Grb2-SOS complex and Ras activation, we observed that insulin stimulation results in a rapid but transient activation of Ras and the extracellular-signal regulated kinase (ERK) followed by dissociation of the Grb2-SOS complex. Although treatment with the phorbol myristate acetate resulted in ERK activation and complete dissociation of the Grb2-SOS complex, there was no effect on subsequent insulin-stimulated Ras activation. Similarly, insulin stimulation followed by insulin removal resulted in a time-dependent restoration of the Grb2-SOS complex but which was significantly slower than the recovery of insulin-stimulated Ras activation. In addition, although insulin was able to activate Ras under these conditions, there was a complete desensitization of Raf and ERK activation. This apparent homologous desensitization of insulin action was specific for Raf and ERK as the insulin re-stimulation of insulin receptor autophosphorylation and protein kinase B activation were unaffected. Together, these data demonstrate the presence of a pathway independent of the Grb2-SOS complex that can lead to Ras activation but that the desensitization of Raf accounts for the homologous desensitization of ERK.     

Cytologic features based on the expression of E-cadherin and catenins in lung adenocarcinoma.

OBJECTIVE: To investigate the relationship between E-cadherin-associated cell-to-cell adhesion and cytologic features in preoperative cytologic lung adenocarcinoma specimens. STUDY DESIGN: Evaluation of the relationship between cell-to-cell adhesion, formation of cellular clusters and frequency of single cells in 31 cases of primary lung adenocarcinoma, collected by brush and needle cytology preoperatively. RESULTS: Most cases with remarkable overlapping of cells in compact cellular clusters and a few solitary cells maintained cell-to-cell adhesion. Cellular clusters that had a slight tendency to overlap, a small cell-to-cell adhesion area and a high frequency of solitary cells tended to lack E-cadherin-associated cell-to-cell adhesion. CONCLUSION: Formation of cellular clusters and the appearance of solitary cancer cells are closely related to E-cadherin-associated cell-to-cell adhesion. Therefore, it is highly likely that cytologic features may indicate malignant behavior, such as local invasion and lymph node metastasis, in primary lung adenocarcinoma.     

Isoprenoid quinone, cellular fatty acid composition and diaminopimelic acid isomers of newly classified thermophilic anaerobic Gram-positive bacteria

The configuration of quinone systems, cellular fatty acids and diaminopimelic acids in 11 species of thermophilic clostridia which have been classified into new genera based on their 16S rRNA sequences was determined. It was found that menaquinone 7 was present in 10 species as the major component of menaquinone systems by analyses using HPLC with photodiode-array detector and electron impact mass spectrometry. In seven of the 11 species, the major cellular fatty acids were determined to be iso-15:0 and iso-17:0. As a results of chemotaxonomic studies it was demonstrated that the representatives of the new genera Thermoanaerobacter and Thermoanaerobacterium were heterogeneous in their chemical composition, whereas strains of the new genus Moorella and of the new unnamed genus which is composed of (Clostridium) stercorarium and (Clostridium) thermolacticum showed homogeneity in their chemotaxonomic characteristics.     

In vitro and in vivo survival of frozen-thawed bovine oocytes after IVF,nuclear transfer,and parthenogenetic activation

Cryopreservation of bovine oocytes would be beneficial both for nuclear transfer and for preservation efforts. The overall objective of this study was to evaluate the viability as well as the cryodamage to the nucleus vs. cytoplasm of bovine oocytes following freezing-thawing of oocytes at immature (GV) and matured (MII) stages using in vitro fertilization (IVF), parthenogenetic activation, or nuclear transfer assays. Oocytes were collected from slaughterhouse ovaries. Oocytes at the GV, MII, or MII but enucleated (MIIe) stages were cryopreserved in 5% (v/v) ethylene glycol; 6% (v/v) 1,2-propanediol; and 0.1-M sucrose in PBS supplemented with 20% (v/v) fetal bovine serum. Frozen-thawed oocytes were subjected to IVF, parthenogenetic activation, or nuclear transfer assays. Significantly fewer GV oocytes survived (i.e., remained morphologically intact during freezing-thawing) than did MII oocytes (47% vs. 84%). Subsequent development of the surviving frozen-thawed GV and MII oocytes was not different (58% and 60% cleavage development; 7% and 12% blastocyst development at Day 9, respectively, P > 0.05). Parthenogenetic activation of frozen-thawed oocytes resulted in significantly lower rates of blastocyst development for the GV than the MII oocyte groups (1% vs. 14%). Nuclear transfer with cytoplasts derived from frozen-thawed GV, MII, MIIe, and fresh-MII control oocytes resulted in 5%, 16%, 14%, and 17% blastocyst development, respectively. However, results of preliminary embryo transfer trials showed that fewer pregnancies were produced from cloned embryos derived from frozen oocytes or cytoplasts (9%, n = 11 embryos) than from fresh ones (19%, n = 21 embryos). Transfer of embryos derived by IVF from cryopreserved GV and MII oocytes also resulted in term development of calves. Our results showed that both GV and MII oocytes could survive freezing and were capable of developing into offspring following IVF or nuclear transfer. However, blastocyst development of frozen-thawed oocytes remains poorer than that of fresh oocytes, and our nuclear transfer assay suggests that this poorer development was likely caused by cryodamage to the oocyte cytoplasm as well as to the nucleus. Mol. Reprod. Dev. 51:281–286, 1998. © 1998 Wiley-Liss, Inc.     

Effects of maternal uninephrectomy on the development of fetal rat kidney: Numerical and volumetric changes of the glomerulus and formation of the anionic site in the glomerular basement membrane

Developmental processes of the fetal rat kidney from uninephrectomized mothers were studied. The uninephrectomy was performed on day 5 of gestation. Glomerular number and volume in the fetal kidneys on days 18, 20, and 22 of gestation were morphometrically determined. To investigate the anionic site formation in the glomerular basement membrane, distribution of cationized ferritin (CF) in the fetal glomerulus was examined electron microscopically after CF injection. Blood urea nitrogen (BUN) concentration in the pregnant rats was also determined on various days after uninephrectomy. On fetal days 20 and 22, the glomerular volume was significantly larger in the fetuses from uninephrectomized mothers than in those from sham-operated ones. On fetal day 20, the CF particles were clustered in the laminae rarae interna and externa of the glomerular basement membrane in the fetuses from uninephrectomized mothers, while the clusters were arrayed in three to four layers in the glomerular basement membrane in the fetuses from sham-operated ones. On fetal day 22, the CF particles noted in the lamina rara externa in the fetuses from uninephrectomized mothers were slightly larger in number than such particles in the age-matched control fetuses. The BUN concentration of the uninephrectomized pregnant rats was significantly higher than that of the sham-operated pregnant ones on each postoperative day. These results suggest that the development of the fetal renal glomerulus is accelerated by the elevated BUN level following maternal uninephrectomy when the fetal kidney is functional in effective filtration in the rat. J. Morphol. 238:337–342, 1998. © 1998 Wiley-Liss, Inc.