[ANXA8 Regulates Proliferation of Human Non-Small Lung Cancer Cells A549 via EGFR-AKT-mTOR Signaling Pathway].

Annexin A8 (ANXA8) is a member of the annexin family, which had been reported to regulate multiple cancer cellular processes including proliferation, metastasis and inflammation. However, the specific role of ANXA8 in lung cancer cell biology remains unknown. Our previous transcriptome study revealed that ANXA8 mRNA was downregulated in curcumin analog (MHMD) -treated human non-small lung cancer cells (A549 cell line). Here, we continued to study the ANXA8 expression in A549 cells using reverse transcription-quantitative PCR and Western blotting, compared with that in human normal bronchial epithelium cells (BE-AS-2B cell line). Overexpression of ANXA8 via transfection of pEGFP-ANXA8 recombinant vector contributed to the proliferation and migration of A549 cells. Moreover, the cell cycle protein cyclin E1 was upregulated in ANXA8-transfected A549 cells. Knockdown of ANXA8 using an RNA interference technique decreased A549 cell viability and restrained their migration in vitro. The expression levels of multiple cellular factors, including EGFR, PI3K, Akt, mTOR, p70S6K and 4EBP1, in the epidermal growth factor receptor (EGFR) signaling pathway were also altered by ANXA8 knockdown or overexpression in A549 cells, which confirmed the activation of the EGFR/Akt/mTOR signaling pathway by ANXA8. The present results provided evidence to support further investigation of the functional identification of ANXA8 in lung cancer cells in the future.

Long non-coding RNA plasmacytoma variant translocation 1 (PVT1) promotes glioblastoma multiforme progression via regulating miR-1301-3p/TMBIM6 axis.

OBJECTIVE: To explore whether plasmacytoma variant translocation 1 (PVT1) could regulate glioblastoma multiforme (GBM) progression via microRNA-1301-3p (miR-1301-3p) and transmembrane BAX inhibitor motif containing 6 (TMBIM6) axis. MATERIALS AND METHODS: Expression patterns of PVT1 and RMBIM6 in GBM patients were analyzed using GEPIA, an online gene expression analysis tool. Levels of PVT1 in GBM cells and normal cells were analyzed with quantitative real-time PCR method. Cell Counting Kit-8 (CCK-8), transwell invasion assay, and flow cytometry assay were applied to detect cell viability and apoptosis. Connections of PVT1 or TMBIM6 with miR-1301-3p were validated with bioinformatic tool and luciferase activity reporter assay. RESULTS: PVT1 was significantly expressed in GBM tissues and cells. PVT1 promotes GBM cell proliferation and invasion but inhibits apoptosis in vitro. TMBIM6 was significantly expressed in GBM tissues. The knockdown of TMBIM6 reversed the stimulation effects of PVT1 on GBM cell malignancy behaviors with miR-1301-3p as a bridge. CONCLUSIONS: Collectively, we showed PVT1 elevated TMBIM6 expression mediated by miR-1301-3p and thus to promote GBM progression.

[Implement of multimodal navigation-based virtual reality in the needle biopsy of intracranial eloquent lesions].

Objective: To investigate the clinical value of multimodal navigation-based virtual reality (MNVR) in the needle biopsy of intracranial eloquent lesions. Methods: From January 2016 to January 2017, 20 patients with intracranial deep-seated lesions involving eloquent brain areas underwent MNVR-aided needle biopsy at Department of Neurosurgery, People's Liberation Army General Hospital. Preoperatively, MNVR was used to propose and revise the biopsy planning. Intraoperatively, navigation helped trajectory avoid the eloquent structures. Intraoperative MRI (iMRI) was performed to prove the biopsy accuracy and detect the intraoperative complications. Perioperative neurological status, iMRI findings, intraoprative complications, surgical outcome and pathological diagnosis were recorded. Wilcoxon rank-sum test was conducted to compare the preoperative and postoperative neurological scores. Results: MNVR helped revised 45%(9/20) initial biopsy trajectories, which would probably injury the nearby eloquent structures. Navigation helped biopsy trajectories spare the eloquent structures during the operation. No statistical difference was found between postoperative and preoperative neurological status, despite all the lesions were adjacent to eloquent areas. Additionally, 20 patients totally received 21 iMRI scanning. iMRI helped revise incorrect biopsy site in one case and detected intraoperative hemorrhage in another case, both of cases were treated immediately and effectively. No MNVR related adverse events and complications occurred. Conclusions: MNVR-aided needle biopsy of intracranial eloquent lesions is a safe, novel and efficient biopsy modality. This technique is helpful to reduce the incidence of surgery related neurological deficits.

[Implementation of multimodal navigation combined with intraoperative magnetic resonance imaging in the frameless stereotactic biopsy of intracranial lesions].

Objective: To investigate the impact and value of multimodal navigation and intraoperative magnetic resonance imaging (iMRI) on the biopsy of intracranial lesions. Methods: From February, 2009 to December, 2016, this study enrolled 156 patients, who underwent multimodal navigation and iMRI-guided brain biopsy in the Neurosurgery Department of PLA General Hospital. Metabolic information was used for biopsy target selection. Intraoperative guidance helped biopsy trajectory avoid the eloquent structures. iMRI was performed to prove the biopsy accuracy and to revise the incorrect biopsy. Diagnostic rate, perioperative neurological status, surgical parameter, and surgical outcome were recorded. Results: The first iMRI helped to revise 7 (4.5%) incorrect biopsy sites, and final iMRI confirmed biopsy accuracy in all cases. Postoperative diagnostic rate was 96.8% (151/156). No statistical difference was found between postoperative and preoperative neurological statuses, despite 86 (55.1%) lesions were adjacent to eloquent areas. Additionally, iMRI detected 6 (3.8%) intraoperative hematomas that were treated immediately. Conclusions: Brian biopsy with iMRI and multimodal navigation is a safe, accurate and efficient biopsy modality. This technique may help increase the biopsy accuracy with low morbidity and mortality.

A consortium of rhizobacterial strains and biochemical growth elicitors improve cold and drought stress tolerance in rice (Oryza sativa L.).

In the present study, a consortium of two rhizobacteria Bacillus amyloliquefaciens Bk7 and Brevibacillus laterosporus B4, termed 'BB', biochemical elicitors salicylic acid and ß-aminobutyric acid (SB) and their mixture (BBSB) were investigated for cold and drought stress tolerance in rice plants. After withholding water for 16 days, rice plants treated with BBSB showed 100% survival, improved seedling height (35.4 cm), shoot number (6.12), and showed minimum symptoms of chlorosis (19%), wilting (4%), necrosis (6%) and rolling of leaves. Similarly, BB inoculation enhanced plant growth and reduced overall symptoms in rice seedlings subjected to 0 ± 5 °C for 24 h. Our results imply several mechanisms underlying BB- and BBSB-elicited stress tolerance. In contrast to the control, both treatments significantly decreased leaf monodehydroascorbate (MDA) content and electrolyte leakage, and increased leaf proline and cholorophyll content. Moreover, activities of antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT) increased 3.0- and 3.6-fold, respectively. Moreover, expression of OsMYB3R-2, OsDIL, OsDREB1A and OsCDPK13 genes was significantly up-regulated, suggesting that these genes play important roles in abiotic stress tolerance of rice. In addition, bacterial strains Bk7 and B4 were able to produce high amounts of IAA and siderophores, and colonise the plant roots, while only strain Bk7 exhibited the capability to form biofilms and solubilise inorganic phosphate. This study indicates that the BB and BBSB bio-formulations can be used to confer induced systematic tolerance and improve the health of rice plants subject to chilling and drought stress.

Synthesis and antimicrobial activity of 9-o-substituted palmatine derivatives.

A series of new palmatine derivatives with alkyl or alkyl with N-heterocyclic structures were designed and synthesized at C-9-O according to the principle of association. These compounds were characterised by (1)H NMR, (13)C NMR, ESI-MS and elemental analysis, and tested for their antimicrobial activity in vitro to evaluate structure-activity relationships. The results indicated that 9-O-substituted palmatine derivatives exhibit varying degrees of antimicrobial activity. Antibacterial activities of compounds (3a-f) against Gram +ve bacteria increased 2- to 64-fold than that of palmatine. The compounds (3a-f) possessed relatively weaker inhibitory effects against Gram -ve bacteria and fungi than that against Gram +ve bacteria. Antimicrobial activities of compounds (5a-e) are lower than that of compounds (3a-f). Compound 3d showed the highest antimicrobial activity of all the compounds. The LD50 values of compounds (3a-f) decreased as the alkyl side chain was elongated. Compound 3f showed least toxicity.

Mechanism of curcumin analog MHMD-induced cell death in A549 lung cancer cells.

OBJECTIVE: To investigate the anticancer properties of a chemosynthetic curcumin analog, (1E,6E)-4-((furan-2-yl)methylene)-1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione (C26H22O7, abbreviated MHMD) in A549 cells. MATERIALS AND METHODS: Inverted microscope was used to observe the alteration on cytomorphology. MTT assay was used to detect cell viability. Acridine-orange staining was used to measure autophagy, and AnnexinV/PI staining and Hoechst/PI staining to measure apoptosis and necrosis. RESULTS: MTT assays showed that at 12 h, 24 h, 48 h, MHMD reduced cell viability with an IC50 of 27.46 µM, 18.86 µM, and 11.23 µM, respectively. Typical characteristics were observed in concert with cell death, including treated-cells getting brighter, rounder, and becoming non-adherent gradually. Additionally, acridine-orange staining suggested that autophagy didn't involve in MHMD-induced cell death. However, apoptosis and necrosis played important roles in MHMD-induced cell death by Hoechst33342/PI staining. It showed apoptosis was the main cause at low concentrations (≤ 4 µM), while with the concentrations rising, necrosis was the leading role. AnnexinV/PI staining again indicated the occurrence of apoptosis at 4 µM. Furthermore, the caspases inhibitor z-VAD-fmk could prevent MHMD-induced cell death, which showed much higher cell viability than those only treated with MHMD (4 µM). Moreover, MTT assay also demonstrated that MHMD did possess a greater anti-proliferative ability than curcumin. CONCLUSIONS: The curcumin analog MHMD is able to induce A549 cell death in a time and dose-dependent manner via apoptosis and necrosis. And MHMD could be a more effective drug than curcumin.

Identification and characterization of rhizosphere fungal strain MF-91 antagonistic to rice blast and sheath blight pathogens.

AIM: To examine the inhibition effects of rhizosphere fungal strain MF-91 on the rice blast pathogen Magnaporthe grisea and sheath blight pathogen Rhizoctonia solani. METHODS AND RESULTS: Rhizosphere fungal strain MF-91 and its metabolites suppressed the in vitro mycelial growth of R. solani. The inhibitory effect of the metabolites was affected by incubation temperature, lighting time, initial pH and incubation time of rhizosphere fungal strain MF-91. The in vitro mycelial growth of M. grisea was insignificantly inhibited by rhizosphere fungal strain MF-91 and its metabolites. The metabolites of rhizosphere fungal strain MF-91 significantly inhibited the conidial germination and appressorium formation of M. grisea. Moreover, the metabolites reduced the disease index of rice sheath blight by 35·02% in a greenhouse and 57·81% in a field as well as reduced the disease index of rice blast by 66·07% in a field. Rhizosphere fungal strain MF-91 was identified as Chaetomium aureum based on the morphological observation, the analysis of 18S ribosomal DNA internal transcribed spacer sequence and its physiological characteristics, such as the optimal medium, temperature and initial pH for mycelial growth and sporulation production. CONCLUSIONS: Rhizosphere fungus C. aureum is effective in the biocontrolling of rice blast pathogen M. grisea and sheath blight pathogen R. solani both in in vitro and in vivo conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first to show that rhizosphere fungus C. aureum is a potential fungicide against rice blast and sheath blight pathogens.

First Report of Anthracnose of Digitaria sanguinalis Caused by Colletotrichum hanaui in China.

Hairy crabgrass (Digitaria sanguinalis (L.) Scop.) is a troublesome weed in most agricultural crops worldwide. Considerable efforts are made to limit the invasiveness and impact of crabgrass on crop productivity, including evaluation of fungi as biocontrol agents (3). In September 2005, a severe disease was observed on crabgrass plants in Zhejiang Province. Leaves and stems of the affected plant showed small, water-soaked, brownish spots that rapidly turned into longitudinal elliptic or spindle-shaped lesions, 6.5 to 8 × 22 to 24 mm, with a brown outer edge and a gray sunken central area. Coalescence of large lesions gave rise to extensive rotting and necrosis, and the stems were broken when the lesions encircled. Acervuli with brown setae and falcate single-celled spores, typical of some Colletotrichum species (2), formed on the lesions at this late stage. One fungal isolate (Col-68) was obtained from symptomatic tissues on potato dextrose agar that led to white-to-gray appressed mycelium growth with orange conidial masses at 28°C in darkness. Setae were septate, dark brown, rounded and sometimes lobed at base, 32.0 to 116.5 × 3.2 to 6.0 µm, with apices acute. Hyphae were septate, hyaline, 1.0 to 6.5 µm, and sometimes guttulate. Conidia were falcate or fusiform, apices acute or obtuse, and 8.16 to 26.37 × 2.9 to 9.2 µm with an average of 18.15 × 5.65 µm. Hyphopodial appressoria were smooth, globose to prolate, ovoid or obovoid with obtuse or cylindrical apices, edges entire, and 4.17 to 14.25 × 3.77 to 8.94 µm with an average of 7.0 × 6.9 µm. The pathogen was initially identified as a Colletotrichum species based on morphology. Suspensions of 3-day-old spores collected from potato dextrose liquid cultures (106 conidia per ml) were used to spray inoculate (15 ml per pot) three 9-cm-diameter pots of crabgrass seedlings at the three- to four-leaf growth stage. Another three pots of healthy crabgrass were simultaneously sprayed with sterilized distilled water without conidia, which served as noninoculated checks. The seedlings were kept at 25 to 28°C for 24 h under a polyethylene sheet cover in the greenhouse. Symptoms that developed in all inoculated seedlings were identical to those observed on the affected crabgrass in the field, meanwhile the seedlings inoculated with sterilized water had no significant symptoms, and the reisolated strain had the same characteristics as the original isolate. To diagnose the pathogen to the species level, three isolates were tested and an approximately 580-bp DNA amplicon of this isolate was amplified using the primers ITS1/ITS4. The sequence (GenBank Accession No. GQ456160) had 98% sequence identity with the sequences of Colletotrichum hanaui (GenBank Accession Nos. EU554101and EU554124), which is supported by phylogenetic analysis with bootstrap support. On the basis of the morphological, pathological characteristics, and phylogenetic tree, the isolated strain was identified as C. hanaui (1). To our knowledge, this is the first confirmed report of anthracnose of D. sanguinalis caused by newly described C. hanaui in China. References: (1) J. A. Crouch et al. Mycologia, 101:717, 2009. (2) B. C. Sutton. The Coelomycetes. CAB International Publishing, New York, 1980. (3) Y. Z. Zhu and S. Qiang. Chin. J. Biol. Control 20:206, 2004.

Effects of age and gender on intravenous midazolam premedication: a randomized double-blind study.

BACKGROUND: Given the potentially important effects that age and gender may have on midazolam premedication, this study aimed at determining if these factors alter anxiety, sedation, and cardiorespiratory outcomes when administering two different doses of i.v. midazolam. METHODS: After randomization, patients were premedicated 1 h before surgery with either i.v. midazolam 0.02 or 0.06 mg kg(-1) depending on their age and gender group. Levels of anxiety and sedation, heart rate, respiratory rate (RR), mean blood pressure (MBP), and oxygen saturation (Sp(O2)) were measured before and 15 min after midazolam administration. RESULTS: A higher level of preoperative anxiety was more often observed in women than in men, and in young than in older patients. The female or younger patients showed significant anxiolytic benefits from midazolam. A deeper sedation level was found in men compared with women. Forty-two of 45 patients (93.3%) with excessive sedation received midazolam 0.06 mg kg(-1). The elderly patients receiving midazolam 0.06 mg kg(-1) showed significant reductions in MBP, RR, and Sp(O2). Of the patients with an Sp(O2)<90%, 72.7% had received midazolam 0.06 mg kg(-1). CONCLUSIONS: Age and gender differences in neuropsychological and physiological responses after midazolam premedication were evident. Midazolam is effective for producing sedation and anxiolysis at a dose of 0.02 mg kg(-1), with minimal effects on cardiorespiration and oxygen saturation to patients. Dosage adjustments based on these covariates are, therefore, necessary.

Administration of donor-derived mesenchymal stem cells can prolong the survival of rat cardiac allograft.

BACKGROUND: Mesenchymal stem cells (MSCs) are multipotent adult elements that have recently been shown to have profound immunomodulatory effects both in vitro and in vivo. Herein we have examined the impact of intravenous infusion of donor MSCs on the survival of transplanted hearts in a rat allograft model. METHODS: Recipient Fisher344 rats were transplanted with hearts from inbred Wistar rats. Wistar rat MSCs were infused via the tail vein at designated intervals. In vitro mixed lymphocyte reaction (MLR) and cell-mediated lympholysis (CML) assays were performed to assess whether MSCs downregulated T-cell responses in vivo. Real-time polymerase chain reaction (PCR) was used to analyze the Th1/Th2 balance in MSC-treated and control groups. RESULTS: The MSCs cultured in vitro exhibited multipotential for differentiation. Survival of the allografts was markedly prolonged by administration of MSCs compared with the controls, namely mean survivals of 12.4 vs 6.4 days, respectively. Real-time PCR showed a shift in the Th1/Th2 balance toward Th2. By MLR and CML assays, untreated control rats showed greater alloreactivity than did MSC-treated rats. CONCLUSION: Our results indicated that MSCs suppressed allogeneic T-cell responses both in vitro and in vivo. Intravenous administration of MSCs prolonged the survival of transplanted hearts, possibly by induction of allograft tolerance through changing the Th1/Th2 balance.

Carbon dioxide laser induced airway fire during larynx surgery: case report.

The precision intrinsic hemostatic properties of the laser have led to its wide use in modern clinical medicine especially in microscopic airway surgery. However, the intense heat generated by the high energy density of the surgical laser can convert combustible tubes into veritable torches, cause catastrophic fires, and result in severe injury to the patient. This is of particular importance when high energy is used on the continuous mode or when the endotracheal tube is repeatedly hit by the laser at the same spot. Most reported laser-induced complications result from the laser beam inadvertently falling on the areas that are not intended to be exposed. We report a case of a trans-tracheostomy tube fire occurring during carbon dioxide (CO2) laser surgery. Aluminum-tape wrapping did not prevent this complication. It was found that the ignition of a trans-tracheostomy tube was caused by the laser striking an unprotected portion of the tube during resection of granuloma of the trachea.

Prolonged interference of patent blue on pulse oximetry readings.

BACKGROUND: We evaluated the effect of intra-arterial injection of Patent Blue (PB) on pulse oximetry. METHODS: Ten consecutive female patients who underwent intra-arterial insertion of catheter for chemotherapy were enrolled in this study. All patients received general anesthesia and ECG, pulse oximetry, and direct arterial blood pressure were monitored throughout the perioperative period. Baseline arterial blood saturation (SpO2), haemoglobin concentration (Hgb), estimated arterial saturation (SaO2), arterial oxygen partial pressure (PaO2) were obtained through arterial blood gas analysis, and true arterial saturation (ScoO2) through CO-Oximetry analysis. The subsequent values obtained at various points of time following the intra-arterial administration of PB were compared with the baseline data. RESULTS: The SpO2 readings taken after intra-arterial injection of PB were significantly lower than the baseline reading. The extent of the reduction of SpO2 readings two hours after the administration of PB was relevant to the total accumulative dosage of PB (rs = -0.846, P < 0.05). However, the reduction of SpO2 bore no significant relation with Hgb concentration or with the ratio of PB dosage to Hgb concentration. CONCLUSIONS: Intra-arterial administration of PB interferes with pulse oximetry readings and the effect lasts for a variable duration. The extent of desaturation may be related to the total accumulative dosage of intra-arterial PB injected.

Isolation and developmental expression of two nuclear receptors, MHR4 and betaFTZ-F1, in the tobacco hornworm, Manduca sexta.

The cDNAs for two members of the nuclear receptor superfamily were isolated from the tobacco hornworm, Manduca sexta. The deduced amino acid sequence of MHR4 shows 93-95% identity in the DNA-binding domain and the first portion of the hinge (D) region with the germ cell nuclear factor (GCNF)-related factors (GRFs) of the silkworm, Bombyx mori, and the mealworm, Tenebrio molitor, and with a genomic sequence from the fruit fly, Drosophila melanogaster. Northern blot hybridization showed that a 7.5 kb MHR4 mRNA appeared in Manduca abdominal epidermis just as the ecdysteroid titer began to decline during the larval molt, disappeared about 12 h later, then transiently reappeared shortly before larval ecdysis. During the pupal and adult molts, a similar pattern of expression was seen (the very end of the adult molt was not studied). At peak times of expression in the epidermis, MHR4 mRNA was also present in fat body and the central nervous system (CNS). The deduced amino acid sequence of Manduca FTZ-F1 is 100% and 96% identical to that of B. mori and Drosophila betaFTZ-F1, respectively, in the DNA-binding domain and the adjacent hinge region including the FTZ-F1 box. Northern blot analysis showed that the >9.5 kb betaFTZ-F1 mRNA appeared in Manduca epidermis during the decline of the ecdysteroid titer in the larval, pupal and adult molts as the first peak of MHR4 mRNA declined, then it disappeared in the larval and pupal molts before the second peak of MHR4 appeared. betaFTZ-F1 mRNA was also found in fat body and the CNS at the time of peak expression in the epidermis during the larval and pupal molts. Both MHR4 and betaFTZ-F1 mRNAs were found in the testis during the onset of spermatogenesis in the prepupal period.

Cutaneous blood vessels in scent pigs.

The purpose of this study was to systematically provide anatomic data for flap research in plastic surgery on the cutaneous blood vessels. Seven scent pigs used in this study were killed anesthetically, and their carotid vessels were intubated and injected with a black liquid rubber. Twenty-four hours later, the integument of the scent pig was removed, and the perforating points of the cutaneous vessels were recorded. The different-sized pieces of integument became transparent. Part of this transparent skin tissue was cut into cross-sectional strips. There were three types of the cutaneous vascular source, the same as in humans. Six division levels of vessels in the skin were identified, which formed five vascular plexuses and two systems (the perforating vessel system and the cutaneous vessel system). There were two sets of vein systems: the concomitant vein and the oscillating vein; the latter can be divided into regular and irregular types. The structures of the perforating vessel system and the cutaneous vessel system were the morphological basis for choosing flaps. Two anatomic points have been emphasized: the preserved vascular plexus in thin flaps (not the subcutaneous vascular network reported previously) and the dependency of vascular structure on its location. Otherwise, this study has also provided two new kinds of flaps used in experimental study: the arterial loop flap and the intermuscular septal perforator flap. Although there were differences as well as similarities in skin vasculature between humans and the scent pig, the scent pig is still suitable for flap research.

109 cases of blepharoptosis treated by forked frontalis muscle aponeurosis procedure with long term follow-up.

109 cases of severe or recurrent blepharoptosis have been treated with the forked frontalis muscle aponeurosis (FFMA) technique since 1989. In comparison with other frontalis muscle flap (FMF) protocols, this technique has three advantages: (i) no skin incision in the lower rim of the eyebrow; (ii) no incision in the frontalis muscle; and (iii) no dissection under the frontalis muscle. The FFMA is formed at the junction of the frontalis and orbicularis muscles. The 9-year follow-up shows that this is a highly effective procedure. The postoperative function of the frontalis muscle is good and the lack of damage has been confirmed by EMG. There are a few complications such as the sluggishness of the upper eyelid on downward gaze and the possibility of asymmetrical brow height in unilateral blepharoptosis. However, this technique may serve as the best choice in the treatment of severe or recurrent blepharoptosis.

Sequences of two cDNAs encoding silkworm homologues of Drosophila melanogaster squid gene.

The squid (sqd) gene of Drosophila melanogaster encodes a few isoforms of a heterogeneous nuclear (hn) RNA-binding protein. We isolated two types of cDNAs coding for homologues of the Sqd protein from the silkworm Bombyx mori. The two predicted amino acid (aa) sequences are identical up to aa 280 and then diverge. The silkworm and fruit fly proteins share 80% homology in the RNA-binding motif region. These cDNAs detect 2.0-, 1.8- and 1-kb mRNAs in the middle and posterior silk glands.

Intermittent expression of BmFTZ-F1, a member of the nuclear hormone receptor superfamily during development of the silkworm Bombyx mori.

BmFTZ-F1 is a sequence-specific DNA-binding factor in the silkworm Bombyx mori sharing similar biochemical characteristics with Drosophila FTZ-F1, a member of the nuclear hormone receptor superfamily. Using DNA sequence homology with FTZ-F1 and information on tryptic peptide sequences of BmFTZ-F1, we isolated a cDNA encoding for BmFTZ-F1. Amino acid sequences in the zinc finger DNA-binding region and the putative ligand-binding domain of BmFTZ-F1 showed strong similarity to not only FTZ-F1 but also its mammalian homologues, LRH-1, ELP, and Ad4BP, suggesting the importance of each region for the function of these proteins. Northern blot analyses of RNA isolated from the middle and posterior silk glands and fat bodies showed that a 6.1-kb BmFTZ-F1 mRNA is present in all tissues so far examined. Expression of BmFTZ-F1 mRNA is intermittent, being high during larval molting and both the larval-pupal and the pupal-adult transformations. Injection of 20-hydroxyecdysone at the third day of the 5th instar larvae induced BmFTZ-F1 mRNA in the posterior silk gland after 24 hr. When 5th instar silk glands were cultured in vitro, BmFTZ-F1 mRNA was induced by a 6-hr exposure to 20-hydroxyecdysone followed by 6 hr in hormone-free medium. These results suggest that BmFTZ-F1 is inducible by decline in the ecdysteroid titer and may play an important role in the development of the silkworm as a transcription factor.

A novel DNA-binding motif abuts the zinc finger domain of insect nuclear hormone receptor FTZ-F1 and mouse embryonal long terminal repeat-binding protein.

Fruit fly FTZ-F1, silkworm BmFTZ-F1, and mouse embryonal long terminal repeat-binding protein are members of the nuclear hormone receptor superfamily, which recognizes the same sequence, 5'-PyCAAGGPyCPu-3'. Among these proteins, a 30-amino-acid basic region abutting the C-terminal end of the zinc finger motif, designated the FTZ-F1 box, is conserved. Gel mobility shift competition by various mutant peptides of the DNA-binding region revealed that the FTZ-F1 box as well as the zinc finger motif is involved in the high-affinity binding of FTZ-F1 to its target site. Using a gel mobility shift matrix competition assay, we demonstrated that the FTZ-F1 box governs the recognition of the first three bases, while the zinc finger region recognizes the remaining part of the binding sequence. We also showed that the DNA-binding region of FTZ-F1 recognizes and binds to DNA as a monomer. Occurrence of the FTZ-F1 box sequence in other members of the nuclear hormone receptor superfamily raises the possibility that these receptors constitute a unique subfamily which binds to DNA as a monomer.