Four DEF-like MADS box genes displayed distinct floral morphogenetic roles in Phalaenopsis orchid

The complex flower organization of orchids offers an opportunity to discover new variant genes and different levels of complexity in the morphogenesis of flowers. In this study, four B-class Phalaenopsis DEF-like MADS-box genes were identified and characterized, including PeMADS2, PeMADS3, PeMADS4 and PeMADS5. Differential expression profiles of these genes were detected in the floral organs of P. equestris, suggesting distinctive roles in the floral morphogenesis of orchids. Furthermore, expressions of these genes were varied to different extents in the peloric mutants with lip-like petals. Expression of PeMADS4 was in lips and columns of wild type, and it extended to the lip-like petals in the peloric mutant. Expression of PeMADS5 was mainly in petals and to a lesser extent in columns in the wild type, whereas it was completely eliminated in the peloric mutant. Disruption of the PeMADS5 promoter region of the peloric mutant was detected at nucleotide +312 relative to the upstream of translational start codon, suggesting that a DNA rearrangement has occurred in the peloric mutant. Genomic structure analysis of the PeMADS5 showed that the exon length was conserved in exons 1-6, similar to DEF-like genes of other plants. Collectively, this is the first report that four DEF-like MADS genes were identified in a single monocotyledonous species and that they may play distinctive morphogenetic roles in the floral development of an orchid.     

Free proximal gracilis muscle and its skin paddle compound flap transplantation for complex facial paralysis

Gracilis functioning free-muscle transplantation for the correction of pure facial paralysis has been a preferred method used by many reconstructive microsurgeons. However, for complex facial paralysis, the deficits include facial paralysis along with soft-tissue, mucosa, and/or skin defects. No adequate solution has been proposed. Treatment requests in those patients are not only for facial reanimation but also for correction of the defects. Of 161 patients with facial paralysis treated with gracilis functioning free-muscle transplantation from 1986 to 2002, eight patients (5 percent) presented with complex deficits requiring not only facial reanimation but also aesthetic correction of tissue defects. The tissue defects included an intraoral defect created following contracture release (one patient), infra-auricular radiation dermatitis with contour depression (one patient), temporal depression following a temporalis muscle-fascia transfer (one patient), ear deformity (two patients), and infra-auricular atrophic tissue with contour depression (three patients). A compound flap, consisting of a gracilis muscle with its overlying skin paddle separated into two components, was transferred for simultaneous correction of both problems. The blood supply to the gracilis and to the skin paddle originated from the same source vessel and therefore required the anastomosis of only one set of vessels. The versatility of this compound flap allows for a wide arc of rotation of the skin paddle around the muscle. All flaps were transferred successfully without complications. Satisfactory results of facial reanimation were recorded in five patients after all stages were completed. The remaining three patients are undergoing physical therapy and waiting for revision of the skin paddle.     

Design of general bidirectional associative memories with improved recall capability

The concept of qth order Hamming ball for general bidirectional associative memories (GBAMs) is presented, which results in an enhanced recall capability. A technique based on the minimum overlap algorithm (MOA) is proposed to deal with the separability problem of the qth order Hamming balls. Simulation results show that the recall capability obtained by using the proposed method is indeed larger than that obtained by using the conventional method.     

Acidic-rich region of amyloid precursor protein induces glial cell apoptosis

Amyloid precursor protein (APP) has several caspase cleavage sites in its C-terminal cytoplasmic domain and N-terminal extracellular domain. Caspase cleavages of APP at its cytosolic tail may result in releasing the domain and inducing cell death. During apoptosis, the N-terminal domain may also be processed at amino acids 197 and 219 by caspases leading to unmasking of an acidic-rich region (AR). In this study, AR-exposing APP was shown to inhibit cell growth after transfection into RBA-1 astrocytes and BV-2 microglial cells. The recombinant AR from residue 220 to 288 of APP (APP220-288) was produced and its biological activities were analyzed. APP220-288 induced morphological changes, cell death, and DNA fragmentation in BV-2 and RBA-1 cells. However, AR was determined to have no apparent effects in suspension cells, erythroleukemia K562 cells, and Jurkat T cells. The cytotoxicity was depending on negative charge cluster and the apoptotic activity of AR was attributed to the inhibition of cell adhesion. In BV-2 microglial cells, AR significantly stimulated Fas expression, although expressions of the pro-inflammatory cytokine genes were not detected. APP220-288 also induced nitric oxide synthase (iNOS) expression and nitric oxide (NO) production. These findings indicate that the acidic-rich domain of APP may have apoptotic activity due to inhibition of cell adhesion and induction of iNOS and Fas expressions. Moreover, unmasking the apoptosis-induced AR may activate and exacerbate glial cells which in turn lead to further progression of the death program.     

Intramuscular electroporation with the pro-opiomelanocortin gene in rat adjuvant arthritis

Endogenous opioid peptides have an essential role in the intrinsic modulation and control of inflammatory pain, which could be therapeutically useful. In this study, we established a muscular electroporation method for the gene transfer of pro-opiomelanocortin (POMC) in vivo and investigated its effect on inflammatory pain in a rat model of rheumatoid arthritis. The gene encoding human POMC was inserted into a modified pCMV plasmid, and 0-200 microg of the plasmid-POMC DNA construct was transferred into the tibialis anterior muscle of rats treated with complete Freund's adjuvant (CFA) with or without POMC gene transfer by the electroporation method. The safety and efficiency of the gene transfer was assessed with the following parameters: thermal hyperalgesia, serum adrenocorticotropic hormone (ACTH) and endorphin levels, paw swelling and muscle endorphin levels at 1, 2 and 3 weeks after electroporation. Serum ACTH and endorphin levels of the group into which the gene encoding POMC had been transferred were increased to about 13-14-fold those of the normal control. These levels peaked 1 week after electroporation and significantly decreased 2 weeks after electroporation. Rats that had received the gene encoding POMC had less thermal hypersensitivity and paw swelling than the non-gene-transferred group at days 3, 5 and 7 after injection with CFA. Our promising results showed that transfer of the gene encoding POMC by electroporation is a new and effective method for its expression in vivo, and the analgesic effects of POMC cDNA with electroporation in a rat model of rheumatoid arthritis are reversed by naloxone.     

Cytotoxicity of phenylpropanoid esters from the stems of Hibiscus taiwanensis

The separation of an extract prepared from the stems of the previously uninvestigated Hibiscus taiwanensis led to the isolation of three new phenylpropanoid esters, (7S,8S)-demethylcarolignan E (1), hibiscuwanin A (2), hibiscuwanin B (3), in addition to eight known ones. The structures of these compounds were elucidated by spectroscopic and chemical transformation studies. In cytotoxicity evaluation of the isolates, 9,9'-O-feruloyl-(-)-secoisolaricinresinol (8) showed strong cytotoxic activity against human lung carcinoma and breast carcinoma cell lines in an in vitro cytotoxicity assay with EC(50) values of 1.8 and 3.9 microg/mL, respectively.     

Arginine vasopressin produces inhibition upon respiration without pressor effect in the rat

The purpose of the current study was to examine where arginine vasopressin (AVP) inhibits respiration by direct action on the areas of the ventrolateral medulla (VLM) in the rat. The animal was anesthetized by urethane (1.2 g/kg, i.p.), paralyzed with gallamine triethiodide, and artificially ventilated. Catheterization of the femoral artery and vein, and bilateral vagotomy were performed. The rat was then placed upon a stereotaxic instrument in a prone position. The phrenic nerve was separated and cut peripherally. Phrenic nerve activity (PNA) was monitored at normocapnia and hypercapnia in hyperoxia. Microinjection of AVP into various subregions of the VLM was then performed. In response to AVP microinjection, a transient period of apnea and then a significant decrease in PNA amplitude were observed. Arterial blood pressure was unchanged. This inhibition of PNA with AVP treatment was site-specific, attenuated by raising CO2 concentration, and totally abolished by pretreatment with AVP V1A receptor antagonist. Data of the present study indicate that endogenous resource of AVP may produce an inhibitory effect upon respiration via AVP receptors presented on neurons within the VLM.     

Mechanism of cooperative effects of rhinovirus and atopic sensitization on airway responsiveness

To elucidate the mechanistic interplay between rhinovirus (RV) exposure and atopic sensitization in regulating airway smooth muscle (ASM) responsiveness, isolated rabbit ASM tissue and cultured human ASM cells were passively sensitized with sera from atopic asthmatic or nonatopic nonasthmatic (control) subjects in the absence and presence of inoculation with RV serotype 16. Relative to control subjects, atopic asthmatic serum-sensitized and RV-inoculated ASM exhibited significantly increased contractility to acetylcholine, impaired relaxation to isoproterenol, and enhanced release of the proinflammatory cytokine interleukin-1beta. These effects were potentiated in atopic asthmatic serum-sensitized ASM concomitantly inoculated with RV and inhibited by pretreating the tissues with monoclonal blocking antibodies against intercellular adhesion molecule (ICAM)-1 (CD54), the host receptor for RV serotype 16, or lymphocyte function-associated antigen (LFA)-1 (CD11a/CD18), the endogenous counterreceptor for ICAM-1. Moreover, RV inoculation was found to potentiate the induction of mRNA and surface protein expression of FcepsilonRII (CD23), the low-affinity receptor for IgE, in atopic asthmatic serum-sensitized ASM. Collectively, these observations provide new evidence demonstrating that 1) RV exposure and atopic sensitization act cooperatively to potentiate induction of proasthmatic changes in ASM responsiveness in association with upregulated proinflammatory cytokine release and FcepsilonRII expression and 2) the effects of RV exposure and atopic sensitization are mediated by cooperative ICAM-1-coupled LFA-1 signaling in the ASM itself.     

Hexasaccharides from the histamine-modified depolymerization of porcine intestinal mucosal heparin

Specific sequences in heparin are responsible for its modulation of the biological activity of proteins. As part of a program to characterize heparin-peptide and heparin-protein binding, we are studying the interaction of chemically discrete heparin-derived oligosaccharides with peptides and proteins. We report here the isolation and characterization, by one- and two-dimensional 1H NMR spectroscopies, of ten hexasaccharides, one pentasaccharide, and one octasaccharide serine that were isolated from depolymerized porcine intestinal mucosal heparin. Hexasaccharides were chosen for study because they fall within the size range, typically tetra- to decasaccharide in length, of heparin sequences that modulate the activity of proteins. The depolymerization reaction was catalyzed by heparinase I (EC 4.2.2.7) in the presence of histamine, which binds site specifically to heparin. Histamine increases both the rate and extent of heparinase I-catalyzed depolymerization of heparin. It is proposed that oligosaccharides produced by heparinase I-catalyzed depolymerization can inhibit the enzyme by binding to the imidazolium group of histidine-203, which together with cysteine-135 forms the catalytic domain of heparinase I. The increased rate and extent of depolymerization are attributed to competitive binding of the oligosaccharides by histamine.