Structure-activity relationships in flexible protein domains: regulation of rho GTPases by RhoGDI and D4 GDI

The guanine dissociation inhibitors RhoGDI and D4GDI inhibit guanosine 5'-diphosphate dissociation from Rho GTPases, keeping these small GTPases in an inactive state. The GDIs are made up of two domains: a flexible N-terminal domain of about 70 amino acid residues and a folded 134-residue C-terminal domain. Here, we characterize the conformation of the N-terminal regions of both RhoGDI and D4GDI using a series of NMR experiments which include (15)N relaxation and amide solvent accessibility measurements. In each protein, two regions with tendencies to form helices are identified: residues 36 to 58 and 9 to 20 in RhoGDI, and residues 36 to 57 and 20 to 25 in D4GDI. To examine the functional roles of the N-terminal domain of RhoGDI, in vitro and in vivo functional assays have been carried out with N-terminally truncated proteins. These studies show that the first 30 amino acid residues are not required for inhibition of GDP dissociation but appear to be important for GTP hydrolysis, whilst removal of the first 41 residues completely abolish the ability of RhoGDI to inhibit GDP dissociation. The combination of structural and functional studies allows us to explain why RhoGDI and D4GDI are able to interact in similar ways with the guanosine 5'-diphosphate-bound GTPase, but differ in their ability to regulate GTP-bound forms; these functional differences are attributed to the conformational differences of the N-terminal domains of the guanosine 5'-diphosphate dissociation inhibitors. Therefore, the two transient helices, appear to be associated with different biological effects of RhoGDI, providing a clear example of structure-activity relationships in a flexible protein domain.     

Mechanisms for GroEL/GroES-mediated folding of a large 86-kDa fusion polypeptide in vitro

Our understanding of mechanisms for GroEL/GroES-assisted protein folding to date has been derived mostly from studies with small proteins. Little is known concerning the interaction of these chaperonins with large multidomain polypeptides during folding. In the present study, we investigated chaperonin-dependent folding of a large 86-kDa fusion polypeptide, in which the mature maltose-binding protein (MBP) sequence was linked to the N terminus of the alpha subunit of the decarboxylase (E1) component of the human mitochondrial branched-chain alpha-ketoacid dehydrogenase complex. The fusion polypeptide, MBP-alpha, when co-expressed with the beta subunit of E1, produced a chimeric protein MBP-E1 with an (MBP-alpha)2beta2 structure, similar to the alpha2 beta2 structure in native E1. Reactivation of MBP-E1 denatured in 8 M urea was absolutely dependent on GroEL/GroES and Mg2+-ATP, and exhibited strikingly slow kinetics with a rate constant of 376 M-1 s-1, analogous to denatured untagged E1. Chaperonin-mediated refolding of the MBP-alpha fusion polypeptide showed that the folding of the MBP moiety was about 7-fold faster than that of the alpha moiety on the same chain with rate constants of 1.9 x 10(-3) s-1 and 2.95 x 10(-4) s-1, respectively. This explained the occurrence of an MBP-alpha. GroEL binary complex that was isolated with amylose resin from the refolding mixture and transformed Escherichia coli lysates. The data support the thesis that distinct functional sequences in a large polypeptide exhibit different folding characteristics on the same GroEL scaffold. Moreover, we show that when the alpha.GroEL complex (molar ratio 1:1) was incubated with GroES, the latter was capable of capping either the very ring that harbored the 48-kDa (His)6-alpha polypeptide (in cis) or the opposite unoccupied cavity (in trans). In contrast, the MBP-alpha.GroEL (1:1) complex was capped by GroES exclusively in the trans configuration. These findings suggest that the productive folding of a large multidomain polypeptide can only occur in the GroEL cavity that is not sequestered by GroES.     

Dynamic analysis of T-lymphocyte function in relation to hepatopathologic changes and effect of interleukin-12 treatment in mice infected with Schistosoma japonicum

We analyzed the dynamics of splenic T-lymphocyte function in relation to hepatopathologic changes in C3H/Hc mice, experimentally infected with Schistosoma japonicum. Vigorous granuloma formation was observed at 7 wk postinfection. At 10 wk postinfection, granuloma formation entered into the down-modulation stage, as represented by the diminished granuloma size. The Th2 response was activated when eggs appeared in the liver, whereas Th1 responses were depressed and the proliferation of T lymphocytes was decreased. The level of IgG antibodies to the worm and egg antigens rose continually after infection. Interleukin-12 treatment of infected mice inhibited Th2 responses and T-cell proliferation, decreased granuloma formation and fibrosis, but had no effect on the fecundity of the worms. These data suggest that egg deposition is the major factor driving Th2 responses, depressing Th1 cytokine expression as well as T-cell proliferation in S. japonicum-infected mice.     

The super-cooling agent icilin reveals a mechanism of coincidence detection by a temperature-sensitive TRP channel

TRPM8, a member of the transient receptor potential family of ion channels, depolarizes somatosensory neurons in response to cold. TRPM8 is also activated by the cooling agents menthol and icilin. When exposed to menthol or cold, TRPM8 behaves like many ligand-gated channels, exhibiting rapid activation followed by moderate Ca(2+)-dependent adaptation. In contrast, icilin activates TRPM8 with extremely variable latency followed by extensive desensitization, provided that calcium is present. Here, we show that, to achieve full efficacy, icilin requires simultaneous elevation of cytosolic Ca2+, either via permeation through TRPM8 channels or by release from intracellular stores. Thus, two stimuli must be paired to elicit full channel activation, illustrating the potential for coincidence detection by TRP channels. Determinants of icilin sensitivity map to a region of TRPM8 that corresponds to the capsaicin binding site on the noxious heat receptor TRPV1, suggesting a conserved molecular logic for gating of these thermosensitive channels by chemical agonists.     

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.