Arabidopsis transportin1 is the nuclear import receptor for the circadian clock-regulated RNA-binding protein AtGRP7

We characterized the Arabidopsis orthologue of the human nuclear import receptor transportin1 (TRN1). Like the human receptor, Arabidopsis TRN1 recognizes nuclear import signals on proteins that are different from the classical basic nuclear localization signals. The M9 domain of human heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is the prototype of such signals. We show that AtTRN1 binds to similar domains in hnRNP-like proteins from plants. AtTRN1 also interacts with human hnRNP A1 and with yeast Nab2p, two classical import cargo proteins of transportin in these organisms. Like all nuclear transport receptors of the importin-beta family, AtTRN1 binds to the regulatory GTPase Ran from Arabidopsis. We demonstrated that the amino terminus of AtTRN1 is necessary for this interaction. Recombinant AtTRN1 conferred nuclear import of fluorescently labelled BSA-M9 peptide conjugates in permeabilized HeLa cells, functionally replacing human TRN1 in these in vitro nuclear import assays. We identified three plant substrate proteins that interact with AtTRN1 and contain M9-like domains: a novel Arabidopsis hnRNP that shows high similarity to human hnRNP A1 and two small RNA-binding proteins from Arabidopsis, AtGRP7 and AtGRP8. Nuclear import activity of the M9-like domains of these plant proteins was demonstrated in vivo by their ability to confer partial nuclear re-localisation of a GFP fusion protein containing a nuclear export signal. In addition, fluorescently labelled AtGRP7 was specifically imported into nuclei of permeabilized HeLa cells by Arabidopsis AtTRN1 and human TRN1. These results suggest that the transportin-mediated nuclear import pathway is highly conserved between man, yeast and plants.     

A defect in a novel ADAMTS family member is the cause of the belted white-spotting mutation

Several features of the pigment defect in belted (bt) mutant mice suggest that it occurs as a result of a defect in melanocyte development that is unique from those described for other classical white-spotting mutations. We report here that bt mice carry mutations in Adamts20, a novel member of the ADAMTS family of secreted metalloproteases. Adamts20 shows a highly dynamic pattern of expression in the developing embryo that generally precedes the appearance of melanoblasts in the same region, and is not expressed in the migrating cells themselves. Adamts20 shows remarkable homology with GON-1, an ADAMTS family protease required for distal tip cell migration in C. elegans. Our results suggest that the role of ADAMTS proteases in the regulation of cell migration has been conserved in mammalian development.     

The protective effect of lisinopril on membrane-bound enzymes in myocardial preservation

A number of studies have reported that oxidant stress reduces the activity of isolated Na(+)-K(+) ATPase and Ca(2+) ATPase which are known to affect the cell membrane integrity. The aim of the study is to determine whether the administration of lisinopril is able to protect the membrane-bound enzyme levels in isolated guinea pig hearts and also ascertain whether or not a relationship exists between oxygen free radicals and membrane bound Na(+)-K(+) ATPase and Ca(2+) ATPase. Forty guinea pig hearts were studied in an isolated Krebs-Henseleit solution-perfused Langendorff cardiac model. In all groups cardioplegic arrest was achieved by administering St. Thomas' Hospital cardioplegic solution (STHCS). Group 1 (control, n=10) received only STHCS. Group 2 (n=10) were arrested with lisinopril (l micromol l(-1)) added STHCS. Group 3 (n=10) were pretreated with oral lisinopril (0.2 mg kg(-1) twice a day) for 10 days and then arrested with STHCS. Group 4 were also pretreated with oral lisinopril (0.2 mg kg(-1) twice a day for 10 days), arrested with STHCS and reperfused with lisinopril added to Krebs-Henseleit solution (l micromol l(-1)). Hearts were subjected to normothermic global ischaemia for 90 min and then reperfused at 37 degrees C. Pretreatment and addition of lisinopril in the reperfusion buffer improved the levels of membrane-bound enzymes. When the treated groups were compared with control hearts, the best results were achieved in group 4. The Na(+)-K(+) and Ca(2+) ATPase levels increased from 466.38+/-5.99 to 560.12+/-18.02 and 884.69+/-9.13 to 1287.71+/-13.01 nmolPi mg(-1) protein h(-1) respectively (p<0.05). These results suggest that lisinopril protects the cell membrane integrity and lessens free radical-induced oxidant stress.     

Cancer vaccines: single-epitope anti-idiotype vaccine versus multiple-epitope antigen vaccine

In this study, we compared the immunogenicity and tumor-protective activity of anti-idiotypic antibodies mimicking a single tumor-associated epitope and tumor-associated antigen expressing multiple potentially immunogenic epitopes. We focused our study on the colorectal-carcinoma(CRC)-associated antigen GA733 (also known as CO17-1A/KS1-4/KSA/EpCAM). Monoclonal anti-idiotypic antibody (Ab2) BR3E4 was produced against murine anti-CRC mAb CO17-1A (Ab1) in rats. Full-length native GA733 protein was isolated from human tumor cells, and the extracellular domain protein (GA733-2E) was isolated from supernatants of recombinant baculovirus-infected insect cells by immunoafffinity chromatography. The immunomodulatory activity of the Ab2 was compared with that of the antigen, both in rabbits and in mice. Mice, like humans but not rabbits, express a GA733 antigen homologue on some of their normal tissues. Thus, these in vivo models allow the comparison of the immunogenicity of Ab2 and antigen in the presence (mice) and absence (rabbits) of normal tissue expression and immunological tolerance of the GA733 antigen homologue. In rabbits, aluminum-hydroxide(alum)-precipitated native GA733 antigen was superior to alum-precipitated Ab2 in inducing specific humoral immunity. In mice, alum-precipitated recombinant GA733-2E antigen, but not alum-precipitated Ab2, induced specific humoral immunity. However, when the Ab2 was administered to mice in Freund's complete adjuvant, specific humoral immune responses were elicited. Ab2 in complete Freund's adjuvant and GA733-2E in alum were compared for their capacity to induce antigen-specific cellular immunity in mice. Whereas lymphoproliferative responses were obtained with the recombinant antigen only, delayed-type hypersensitivity responses were obtained with both recombinant antigen and Ab2, although these responses were lower than after antigen immunization. The recombinant antigen in alum did not protect mice against challenge with antigen-positive syngeneic murine CRC cells. Similar studies with Ab2 BR3E4 mimicking the CO17-1A epitope were not possible because the tumor cells do not express this epitope after transfection with the human GA733-2 cDNA. However, similar studies with Ab2 mimicking the epitope defined by mAb GA733, which is expressed by the transfected tumor cells, indicated a lack of tumor-protective activity of this Ab2. In contrast, the full-length antigen expressed by recombinant adenovirus inhibited the growth of established tumors in mice. In conclusion, soluble antigen is a more potent modulator of humoral and cellular immune responses than Ab2, both administered in adjuvant. However, for induction of protective immunity, the immunogenicity of the antigen must be further enhanced, e.g., by expression of the antigen in a viral vector. Received: 27 December 1999 / Accepted: 27 January 2000     

Antioxidative metabolism in down syndrome

Down syndrome is the most common cause of mental retardation, affecting 1 in 700–800 liveborn infants. Although numerous biochemical abnormalities accompanying the syndrome have not yet been completely clarified, the antioxidant defense system enzymes have shown to be altered due to increased gene dosage on chromosome 21 and overproduction of superoxide dismutase (SOD-1 or Cu/Zn SOD). The purpose of this study was to investigate the activities of SOD-1 and glutathione peroxidase (GSH-Px) enzymes and the levels of their cofactors zinc (Zn), copper (Cu) and selenium (Se) in plasma of 20 Down syndrome patients. In comparison with age and sex-matched controls (n=15), plasma GSH-Px, SOD, and Cu levels were significantly decreased in the patient group, but Zn and Se concentrations remained unchanged. This study was presented as a poster in 29th Annual Meeting of European Society of Human Genetics held in Genoa in May, 1997.     

Gastroprotective and Antioxidant Effects of Lobaria pulmonaria and Its Metabolite Rhizonyl Alcohol on Indomethacin‐Induced Gastric Ulcer

Two lichen metabolites, rhizonaldehyde ( 1 ) and rhizonyl alcohol ( 2 ), were isolated from the acetone extract of Lobaria pulmonaria by chromatographic methods, and their chemical structures were determined by UV/VIS, IR, and 1D‐ and 2D‐NMR spectroscopic methods. The gastroprotective and in vivo antioxidant activities of extracts of L. pulmonaria and its metabolites, 1 and 2 , were investigated in indomethacin‐induced ulcer models in rats. The gastric lesions were significantly reduced by acetone, hexane, and CHCl₃ extracts, with 75.3–41.5% inhibition. Rhizonyl alcohol ( 2 ) significantly reduced the gastric lesions with an inhibition rate of 84.6–42.8%, whereas rhizonaldehyde ( 1 ) significantly increased the gastric lesions. Antioxidant parameters and myeloperoxidase activities were also evaluated in the gastric tissues of the rats. Indomethacin caused oxidative stress, which resulted in lipid peroxidation in gastric tissues by decreasing the levels of the antioxidants as compared to healthy rat tissues. In contrast to indomethacin, all extracts and rhizonyl alcohol ( 2 ) caused a significant decrease in lipid peroxidation levels and an increase in antioxidant parameters, superoxide dismutase, glutathione peroxidase, and glutathione‐S‐transferase, and reduced glutathione in gastric tissues. The administration of rhizonyl alcohol ( 2 ) also resulted in a decrease in gastric myeloperoxidase activity increased by indomethacin. The gastroprotective effect of rhizonyl alcohol ( 2 ) can be attributed to its antioxidant properties and its suppressing effect on neutrophil infiltration into gastric tissues.     

Attachment,Symptom Severity,and Depression in Medically Unexplained Musculoskeletal Pain and Osteoarthritis: A Cross-Sectional Study

Background

Attachment insecurity relates to the onset and course of chronic pain via dysfunctional reactions to pain. However, few studies have investigated the proportion of insecure attachment styles in different pain conditions, and results regarding associations between attachment, pain severity, and disability in chronic pain are inconsistent. This study aims to clarify the relationships between insecure attachment and occurrence or severity of chronic pain with and without clearly defined organic cause. To detect potential differences in the importance of global and romantic attachment representations, we included both concepts in our study.

Methods

85 patients with medically unexplained musculoskeletal pain (UMP) and 89 patients with joint pain from osteoarthritis (OA) completed self-report measures of global and romantic attachment, pain intensity, physical functioning, and depression.

Results

Patients reporting global insecure attachment representations were more likely to suffer from medically unexplained musculoskeletal pain (OR 3.4), compared to securely attached patients. Romantic attachment did not differ between pain conditions. Pain intensity was associated with romantic attachment anxiety, and this relationship was more pronounced in the OA group compared to the UMP group. Both global and romantic attachment anxiety predicted depression, accounting for 15% and 17% of the variance, respectively. Disability was independent from attachment patterns.

Conclusions

Our results indicate that global insecure attachment is associated with the experience of medically unexplained musculoskeletal pain, but not with osteoarthritis. In contrast, insecure attachment patterns seem to be linked to pain intensity and pain-related depression in unexplained musculoskeletal pain and in osteoarthritis. These findings suggest that relationship-informed focused treatment strategies may alleviate pain severity and psychological distress in chronic pain independent of underlying pathology.     

Mutational definition of binding requirements of an hnRNP-like protein in Arabidopsis using fluorescence correlation spectroscopy

Arabidopsis thaliana glycine-rich RNA binding protein 7 (AtGRP7) is part of a negative feedback loop through which it regulates alternative splicing and steady-state abundance of its pre-mRNA. Here we use fluorescence correlation spectroscopy to investigate the requirements for AtGRP7 binding to its intron using fluorescently-labelled synthetic oligonucleotides. By systematically introducing point mutations we identify three nucleotides that lead to an increased K_d value when mutated and thus are critical for AtGRP7 binding. Simultaneous mutation of all three residues abrogates binding. The paralogue AtGRP8 binds to an overlapping motif but with a different sequence preference, in line with overlapping but not identical functions of this protein pair. Truncation of the glycine-rich domain reduces the binding affinity of AtGRP7, showing for the first time that the glycine-rich stretch of a plant hnRNP-like protein contributes to binding. Mutation of the conserved R⁴⁹ that is crucial for AtGRP7 function in pathogen defence and splicing abolishes binding.