Growth hormone receptor is a target for presenilin-dependent gamma-secretase cleavage

Growth hormone receptor (GHR) is a cytokine receptor superfamily member that binds growth hormone (GH) via its extracellular domain and signals via interaction of its cytoplasmic domain with JAK2 and other signaling molecules. GHR is a target for inducible metalloprotease-mediated cleavage in its perimembranous extracellular domain, a process that liberates the extracellular domain as the soluble GH-binding protein and leaves behind a cell-associated GHR remnant protein containing the transmembrane and cytoplasmic domains. GHR metalloproteolysis can be catalyzed by tumor necrosis factor-alpha-converting enzyme (ADAM-17) and is associated with down-modulation of GH signaling. We now study the fate of the GHR remnant protein. By anti-GHR cytoplasmic domain immunoblotting, we observed that the remnant induced in response to phorbol ester or platelet-derived growth factor has a reliable pattern of appearance and disappearance in both mouse preadipocytes endogenously expressing GHR and transfected fibroblasts expressing rabbit GHR. Lactacystin, a specific proteasome inhibitor, did not appreciably change the time course of remnant appearance or clearance but allowed detection of the GHR stub, a receptor fragment slightly smaller than the remnant but containing the C terminus of the remnant (receptor cytoplasmic domain). In contrast, MG132, another (less specific) proteasome inhibitor, strongly inhibited remnant clearance and prevented stub appearance. Inhibitors of gamma-secretase, an aspartyl protease, also prevented the appearance of the stub, even in the presence of lactacystin, and concomitantly inhibited remnant clearance in the same fashion as MG132. In addition, mouse embryonic fibroblasts derived from presenilin 1 and 2 (PS1/2) knockouts recapitulated the gamma-secretase inhibitor studies, as compared with their littermate controls (PS1/2 wild type). Confocal microscopy indicated that the GHR cytoplasmic domain became localized to the nucleus in a fashion dependent on PS1/2 activity. These data indicate that the GHR is subject to sequential proteolysis by metalloprotease and gamma-secretase activities and may suggest GH-independent roles for the GHR.     

NELIN, a new F-actin associated protein, stimulates HeLa cell migration and adhesion

A new gene (GenBank Accession No. AF114264) was cloned from umbilical vein wall tissue by using RT-PCR. The gene shares high similarity to the gene encoding F-actin binding protein nexilin, so named as NELIN. A clone of 2737bp contains open reading frame of 1344bp extending from 412 to 1755. NELIN was expressed primarily in the heart and skeletal muscle among eight tested normal tissues. Immunofluorescence and immunoprecipitation demonstrated that NELIN product was associated with F-actin. Stable transfection of NELIN into HeLa cells increased the cell migration by 2.17-fold and the adhesion by 1.67-fold, respectively, compared to cells with the empty vector (P<0.05). The results support that NELIN product is an F-actin associated protein and mediates cell motility.     

Nitric oxide is involved in methyl jasmonate-induced defense responses and secondary metabolism activities of Taxus cells

Methyl jasmonate (MeJA), a methyl ester of jasmonic acid (JA), is a well-established signal molecule in plant defense responses and an effective inducer of secondary metabolite accumulation in plant cell cultures such as the valuable anticancer diterpenoid taxol (paclitaxel) in Taxus spp. This work examines the involvement of nitric oxide (NO) in MeJA-induced plant defense responses and secondary metabolism in Taxus chinensis cell cultures. Exogenously supplied MeJA at 100 microM induced rapid production of NO in the Taxus cell cultures, reaching a maximum within 6 h of MeJA supply. Several other responses occurred concomitantly, including the production of hydrogen peroxide (H2O2), and the increases in intracellular malondialdehyde (MDA) content, lipoxygenase (LOX) and phenylalanine ammonium-lyase (PAL) activities. The MeJA-induced H2O2 production was suppressed by an NO donor, sodium nitroprusside (SNP), but enhanced by NO inhibitors, N (omega)-nitro-L-arginine (L-NNA) and 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (PTIO). In contrast, the MeJA-induced MDA, LOX and PAL were all enhanced by the NO donor but suppressed by the NO inhibitors. The NO inhibitors also suppressed MeJA-induced taxol accumulation. These results are suggestive of a role for NO as a signal element for activating the MeJA-induced defense responses and secondary metabolism activities of plant cells.     

Novel 4-amino-furo[2,3-d]pyrimidines as Tie-2 and VEGFR2 dual inhibitors

A novel class of furo[2,3-d]pyrimidines has been discovered as potent dual inhibitors of Tie-2 and VEGFR2 receptor tyrosine kinases (TK) and a diarylurea moiety at 5-position shows remarkably enhanced activity against both enzymes. One of the most active compounds, 4-amino-3-(4-((2-fluoro-5-(trifluoromethyl)phenyl)amino-carbonylamino)phenyl)-2-(4-methoxyphenyl)furo[2,3-d]pyrimidine (7k) is <3 nM on both TK receptors and the activity is rationalized based on the X-ray crystal structure.     

大熊猫初生胎儿及幼仔组织器官的病理学研究

To elucidate the cause of prenatal and early postnatal death in giant panda, pathological studies have been carried out on paraffin-fixed tissue sections from two fetuses and four cubs. The fetuses appeared to have classical atrophic changes in lung and hemorrhage in multiple organs, whereas the cubs showed purulent inflmnmation in various organs, most profound in lung and umbilicus. Localized infusion of bacteria and neutrophils were identified in the focus. Acute enteritis and hepatitis were also observed, as well as purulent encephalitis in one case. Variable degrees of congestion, hemorrhage, denaturalization and putrescence were evident in heart, liver, spleen, kidney, intestines, and lymph nodes. The results indicated that the fetuses died from suffocation whereas the cubs died from infection.     

Mitochondrial DNA evolution in theDrosophila nasuta subgroup of species

Summary TheDrosophila nasuta group consists of about 12 closely related species distributed throughout the Indo-Pacific region. They are of great interest because of their evolutionary idiosyncrasies including little morphological differentiation, the ability to intercross in the laboratory often producing fertile offspring, and substantial chromosomal evolution. Studies of metric traits, reproductive isolation, and chromosomal and enzyme polymorphisms have failed to resolve the phylogeny of the species. We report the results of a survey of the mitochondrial DNA (mtDNA) restriction patterns of the species. The phylogeny obtained is consistent with other available information and suggests thatD. albomicans may represent the ancestral lineage of the group. The amount of polymorphism in local populations (=1.0% per site) is within the typical range observed in other animals, includingDrosophila. The degree of differentiation between species is, however, low: the origin of the group is tentatively dated about 6–8 million years ago. This study confirms the usefulness of mtDNA restriction patterns for ascertaining the phylogeny of closely related species.     

The domain of the Bacillus subtilis DEAD-box helicase YxiN that is responsible for specific binding of 23S rRNA has an RNA recognition motif fold

The YxiN protein of Bacillus subtilis is a member of the DbpA subfamily of prokaryotic DEAD-box RNA helicases. Like DbpA, it binds with high affinity and specificity to segments of 23S ribosomal RNA as short as 32 nucleotides (nt) that include hairpin 92. Several experiments have shown that the 76-residue carboxy-terminal domain of YxiN is responsible for the high-affinity RNA binding. The domain has been crystallized and its structure has been solved to 1.7 Angstroms resolution. The structure reveals an RNA recognition motif (RRM) fold that is found in many eukaryotic RNA binding proteins; the RRM fold was not apparent from the amino acid sequence. The domain has two solvent exposed aromatic residues at sites that correspond to the aromatic residues of the ribonucleoprotein (RNP) motifs RNP1 and RNP2 that are essential for RNA binding in many RRMs. However, mutagenesis of these residues (Tyr404 and Tyr447) to alanine has little effect on RNA affinity, suggesting that the YxiN domain binds target RNAs in a manner that differs from the binding mode commonly found in many eukaryotic RRMs.     

Role of phosphodiesterase in cyclic AMP signaling in cultured rat granulosa cells

Inactivation of the cyclic nucleotide signal in granulosa cells depends on a complex array of cyclic nucleotide phosphodiesterases (PDE). In order to examine the role of PDE in cyclic AMP (cAMP) signaling in granulosa cells, the present study examined the expression of PDE4D proteins and regulation of cAMP-PDE activities in cultured rat granulosa cells. The results of immunoblot analyses showed that two predominant PDE4D subtypes of approximately 80 and 70 kDa appeared when immature rat granulosa cells were treated with FSH. However, these two new subtypes presumed to be PDE4D proteins were not influenced by treatments of DETA/NO, cGMP and PKB inhibitor, LY294002. Immature rat granulosa cells treated with medium alone displayed low cAMP-PDE activity throughout 48 h of culture while those treated with FSH (2 ng.mL-1) showed a marked increase in cAMP-PDE activity between 6 and 12 h of culture, followed by a decline. The findings from the present study indicate that the increased cAMP-PDE activity by FSH is mainly related to the changes of PDE4D protein levels. However, the inhibitory effects of NO on cAMP accumulation in rat granulosa cells are not via the increased cAMP-PDE activity.     

Probing SWI/SNF remodeling of the nucleosome by unzipping single DNA molecules

Chromatin-remodeling enzymes can overcome strong histone-DNA interactions within the nucleosome to regulate access of DNA-binding factors to the genetic code. By unzipping individual DNA duplexes, each containing a uniquely positioned nucleosome flanked by long segments of DNA, we directly probed histone-DNA interactions. The resulting disruption-force signatures were characteristic of the types and locations of interactions and allowed measurement of the positions of nucleosomes with 2.6-base-pair (bp) precision. Nucleosomes remodeled by yeast SWI/SNF were moved bidirectionally along the DNA, resulting in a continuous position distribution. The characteristic distance of motion was approximately 28 bp per remodeling event, and each event occurred with a catalytic efficiency of 0.4 min(-1) per nM SWI/SNF. Remodeled nucleosomes had essentially identical disruption signatures to those of unremodeled nucleosomes, indicating that their overall structure remained canonical. These results impose substantial constraints on the mechanism of SWI/SNF remodeling.