Model studies for isolation of G-quadruplex-forming DNA sequences through a pull-down strategy with macrocyclic polyoxazole

G-quadruplexes (G4s) are non-B DNA structures present in guanine-rich regions of gene regulatory areas, promoters and CpG islands, but their occurrence and functions remain incompletely understood. Thus, methodology to identify G4 sequences is needed. Here, we describe the synthesis of a novel cyclic hepta-oxazole compound, L1Bio-7OTD (1), bearing a biotin affinity-tag as a tool to pull down G4 structures from mixtures of G4-forming and non G4-forming DNA sequences. We confirmed that it could pull down G4s associated with telomeres, bcl-2 gene, and c-kit gene.     

A Glandular Gift in the German Cockroach, Blattella germanica (L.) (Dictyoptera: Blattellidae): The Courtship Feeding of a Female on Secretions from Male Tergal Glands

In the courtship behavior of the German cockroach, the male presents tergal glands to the female and feeds her with glandular secretions to place her in the appropriate precopulatory position. The phagostimulant activity of the secretions was quantitatively examined using the polyethylene glycol film method. The methanol extract of the glands on the eighth tergite induced a potent feeding response in 6-day-old virgin females (EC ₅₀ = 0.0037 male equivalent/40 g PEG spot). However, there was no temporal relation between the feeding response and the sexual receptivity of the females. Moreover, besides virgin females, the extract induced a feeding response in gravid or mated females, males, and the last-instar nymphs. These results strongly suggest that the secretions function as a dietary feeding stimulant in principle but as a courtship pheromone in the context of courtship behavior where the stimulants are offered as a nuptial gift.     

Effect of 13-hydroperoxyoctadecadienoic acid on the supply of arachidonic acid for prostaglandin synthesis from arachidonoyl-CoA mediated by the cytosolic or microsomal acyl-CoA hydrolase in rabbit kidney medulla

The effects of 13-hydroperoxyoctadecadienoic acid (13-HPODE) on the cytosolic or microsomal acyl-CoA hydrolase (ACH) activity in rabbit kidney medulla and on the ACH-mediated prostaglandin (PG) formation from arachidonoyl-CoA (AA-CoA) were examined. 13-HPODE (10, 20, and 50 microM) had no effect on the cytosolic ACH activity but significantly inhibited the activity of the microsomal enzyme (43-57% inhibition). PG formation was measured as follows: AA-CoA (20 nmol) was preincubated with the cytosolic or microsomal fraction (as the source of ACH) in the presence or absence of 13-HPODE for 5 min at 37 degrees C, followed by incubation with the microsomal fraction (as the source of PG-synthesizing enzymes), hydroquinone and reduced glutathione for 5 min at 37 degrees C, and the PGs formed were measured by HPLC, with use of 9-anthryldiazomethane for derivatization. 13-HPODE reduced the PG formation when the microsomal fraction, but not the cytosolic fraction, was used as the source of ACH (10, 20, and 50 microM; 28-55% inhibition). These results suggest that 13-HPODE may modulate PG levels in rabbit kidney medulla by inhibiting the microsomal ACH activity.     

Cytoplasmic domain is not essential for the cell adhesion activities of gicerin, an Ig-superfamily molecule

Gicerin is a cell adhesion molecule in the immunoglobulin (Ig) superfamily and is expressed abundantly during development in the nervous system. It has homophilic cell adhesion activity and also has heterophilic binding activity with NOF (neurite outgrowth factor) and mediates neurite extension. There are two isoforms of gicerin, one with a short (s-gicerin) and the other with a longer cytoplasmic domain (l-gicerin). We have reported that s-gicerin possesses stronger activities than l-gicerin during cell aggregation, in NOF-binding, and in neurite extension. In this study, we established cell lines which expressed a mutant-gicerin whose cytoplasmic domain was deleted and we compared the above three biological activities of the mutant-gicerin with those of s- and l-gicerin. We found that the mutant-gicerin retained all these activities, but the activities were weaker than those of s-gicerin and almost the same as those of l-gicerin. We concluded that the cytoplasmic domain of gicerin is not essential for optimal adhesive activities of gicerin, but might be involved in the regulation of its activities.     

Involvement of gicerin, a cell adhesion molecule, in development and regeneration of oviduct and metastasis of oviductal adenocarcinomas of the chicken

Gicerin is a novel cell adhesion molecule in the immunoglobulin superfamily and has both homophilic adhesion and heterophilic adhesive activity to neurite outgrowth factor (NOF), an extracellular matrix protein in the laminin family. We investigated the possible involvement of gicerin in oviductal development, regeneration, and metastasis of oviductal adenocarcinomas of the chicken. In the oviductal epithelium, gicerin was expressed strongly during development, disappeared after maturation, and reappeared during regeneration. NOF was constitutively expressed in the basement membrane of the epithelium. These molecules were expressed strongly in oviductal adenocarcinomas in both primary and metastatic lesions in the mesentery. An anti-gicerin antibody inhibited the attachment of adenocarcinoma cells to the mesentery in vitro. Many cells migrated from adenocarcinoma tissues on NOF, which were inhibited by an anti-gicerin antibody. These results suggest that gicerin might play a role in oviductal development and regeneration and also in the metastasis of adenocarcinomas.     

Localization of curved DNA and its association with nucleosome phasing in the promoter region of the human estrogen receptor alpha gene

We determined DNA bend sites in the promoter region of the human estrogen receptor (ER) gene by the circular permutation assay. A total of five sites (ERB-4 to -1, and ERB+1) mapped in the 3 kb region showed an average distance of 688 bp. Most of the sites were accompanied by short poly(dA) x poly(dT) tracts including the potential bend core sequence A2N8A2N8A2 (A/A/A). Fine mapping of the ERB-2 site indicated that this A/A/A and the 20 bp immediate flanking sequence containing one half of the estrogen response element were the sites of DNA curvature. All of the experimentally mapped bend sites corresponded to the positions of DNA curvature as well as to nucleosomes predicted by computer analysis. In vitro nucleosome mapping at ERB-2 revealed that the bend center was located 10-30 bp from the experimental and predicted nucleosome dyad axes.     

Monoclonal antibodies against the minimal DNA-binding domain in the carboxyl-terminal region of human immunodeficiency virus type 1 integrase

Integrase of human immunodeficiency virus type 1 (HIVIN) consists of 288 amino acids, and its minimum DNA-binding domain (MDBD) (amino acids [aa] 220 to 270) is required for the integration reaction. We produced and characterized four murine monoclonal antibodies (MAbs) to the MDBD of HIVIN (strain LAI). Immunoblot and enzyme-linked immunosorbent assays with truncated HIVINs showed that those MAbs recognized sequential epitopes within the MDBD (aa 228 to 236, 237 to 252, 253 to 261, and 262 to 270). Their binding to HIVIN inhibited terminal cleavage and strand transfer activities but not disintegration activity in vitro. This collection of MAbs is useful for studying the structure and function of the MDBD by complementing mutational analyses and other biochemical studies.     

Regulatory molecules for coronary expressions of VEGF and its angiogenic receptor KDR in hypoestrogenic middle-aged female rats

We studied the effects of estrogen deprivation and replacement on the protein and gene expression levels molecules that can be considered to be essential for coronary angiogenesis in middle-aged female rats. The animals were subjected to sham operation, ovariectomy, or ovariectomy with estrogen replacement therapy (ERT). Following ovariectomy, protein and gene expressions of vascular endothelial growth factor (VEGF) and its angiogenic receptor (KDR) showed a marked decline in coronary vessels, as determined by immunohistochemistry and in situ hybridization. ERT resulted in restoration of the ovariectomy-induced changes to intact levels. The coronary expression level of basic fibroblast growth factor was unaffected by estrogen deprivation or treatment. The changes in VEGF and KDR expressions were strongly associated with those in endothelial nitric oxide synthase (eNOS) expression in coronary vessels. Moreover, the age- and gender-dependent accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) protein appeared to be a determinant molecule of VEGF expression in middle-aged female rats. We reached a conclusion that the VEGF-KDR system plays a key role in coronary angiogenesis in hypoestrogenic elderly women and is critically regulated by estrogen, eNOS and HIF-1alpha.