Polysaccharides of Dendrobium officinale inhibit TNF-α-induced apoptosis in A-253 cell line.

OBJECTIVE: Our previous study demonstrated that polysaccharides of Dendrobium officinale Kimura et Migo (DP) were capable of enhancing immunomodulation in an experimental model of Sjögren's syndrome, a chronic autoimmune disease mainly affecting the salivary glands. In the present study, we further investigated the protective effect of DP on a human salivary gland cell line A-253 against tumor necrosis factor (TNF)-α-induced apoptosis. MATERIALS: TNF-α (100 U/ml) was used as the stimulus for treating the A-253 cells to induce cellular apoptosis. Nuclear factor-kappa B (NF-κB, p65), phosphorylation of mitogen-activated protein kinases (MAPK), reactive oxygen species (ROS) generation, mitochondrial membrane potential and proapoptotic proteins were examined. A-253 cells were pre-treated with DP for 12 h before TNF-α stimulation. RESULTS: We observed translocation of NF-κB into the nuclei, prolonged MAPK, excessive ROS generation and strongly decreased mitochondrial membrane potential, and subsequently cytochrome C release and caspase-3 activation. However, pre-treatment with DP significantly inhibited the TNF-α-induced apoptotic factors. CONCLUSIONS: Our data suggested the inhibitory effect of DP on TNF-α-induced apoptosis in a human salivary gland cell line. This inhibition indicated potential inference of DP in the initial plasma membrane-bound complex of TNF-α and its receptors.

Anti-acetylcholinesterase activities of traditional Chinese medicine for treating Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive memory loss and cognitive impairment. It is the most common type of dementia in the ageing population due to a severe loss of cholinergic neurons in selected brain area. At present, acetylcholinesterase inhibitors (AChEI) are the first group of drugs approved by the FDA to treat mild to moderate Alzheimer's disease. Most of these drugs such as huperzine and galanthamine are originally isolated from plants. In this study, the AChE inhibitory activities from extracts of Chinese medicinal herbs that have traditionally been prescribed to treat insomnia and brain function disorders were examined in a 96-well plate assay based on Ellman's method. Both ethanol and aqueous extracts of 26 traditional Chinese medicinal herbs were tested. Inhibitory effects were expressed as the percentage of inhibition. For the herbal extracts that were shown to exert a significant inhibition, dose-dependent inhibitory assays were also performed. Ethanol and aqueous extracts of six herbs were found to have high AChE inhibitory activities in a dose-dependent manner. The IC(50) of these herbal extracts on inhibition of AChE are at around 5-85 microm/ml. The results of this study indicate that there is a great potential to search for novel usage of these medicinal herbs for the treatment of AD.

WITHDRAWN: Physiotherapist-led programmes and interventions for rehabilitation of anterior cruciate ligament, medial collateral ligament and meniscal injuries of the knee in adults.

BACKGROUND: Soft-tissue injuries of the knee, mainly involving the anterior cruciate ligament (ACL), the medial collateral ligament (MCL) and menisci, are common and their rehabilitation after non-surgical or surgical treatment often involves intensive and prolonged physiotherapy. OBJECTIVES: To examine the evidence for effectiveness of various physiotherapist-led (or 'directed') rehabilitation programmes, and of various interventions used within these programmes, for rehabilitation of acute or chronic ACL, MCL or meniscal injuries of the knee in adults. SEARCH STRATEGY: We searched the Cochrane Musculoskeletal Injuries Group's specialised register (to June 2001), MEDLINE (from 1966 to August 1999), EMBASE (from 1980 to February 1997), CINAHL (1982 to April 1999), CURRENT CONTENTS (up to March 1999) and reference lists of relevant articles, and consulted colleagues. Date of the most recent search: June 2001. SELECTION CRITERIA: Randomised or quasi-randomised clinical trials evaluating physiotherapist-led rehabilitation programmes, or components of rehabilitation programmes, for the treatment or post-surgical rehabilitation of ACL, MCL or knee meniscal injuries. Excluded were trials investigating electrical stimulation, or various interventions such as cryotherapy, immobilisation braces and continuous passive motion when used in initial or early treatment. Laboratory based trials reporting intermediate outcomes were also excluded. DATA COLLECTION AND ANALYSIS: All trials, judged as fitting the selection criteria by two reviewers, were independently assessed by two reviewers for methodological quality by use of an 11 item checklist. Data were independently extracted by two reviewers. Any disagreement was resolved by discussion. Although quantitative data from most trials are presented, using relative risks or mean differences together with 95 per cent confidence intervals, trial heterogeneity and lack of outcome data prevented meaningful pooling of results from comparable trials. MAIN RESULTS: Thirty-one trials, involving 1545 mainly young and male patients, met the inclusion criteria of the review. Methodological quality was highly variable: allocation concealment and / or assessor blinding were rare, and assessment of outcome was often incomplete and short-term. ACL injury and /or deficiency was the main focus of 18 trials, MCL injury of two trials, meniscal injury of nine trials and a mixture of soft-tissue injuries in the other two trials. The trial comparisons fell into five main categories: rehabilitation programme versus control (6 trials); one rehabilitation programme versus another (6 trials); different timing of rehabilitation (4 trials); one component of a programme versus another (6 trials); supplementary interventions to a programme versus none (9 trials). No trial provided sufficient evidence to establish the relative effectiveness of the intervention(s) under investigation. AUTHORS' CONCLUSIONS: The available evidence for physiotherapist-led rehabilitation of ACL, MCL and meniscal injuries is wide ranging in terms of scope but insufficient to establish the relative effectiveness of the various approaches and methods in current use. There is a need for further research involving good quality, large scale randomised trials with sufficiently long follow-up to fully assess knee function and recovery.

Cloning of the cDNA of alpha-momorcharin: a ribosome inactivating protein.

Using a lambda gt11 cDNA library constructed from the seeds of the bitter melon (Momordica charantia), we have obtained a full length cDNA containing the entire sequence of alpha-momorcharin by immunoscreening. The length of this cDNA is 1044 basepairs long and it consists of an open reading frame coding for a polypeptide of 286 amino acids. The first 23 residues of this polypeptide probably code for a signal sequence. The N-terminal sequence of the deduced protein is exactly identical to that determined by peptide sequencing. The sequence identity between alpha-momorcharin and other ribosome inactivating proteins, such as trichosanthin and ricin A chain, is high, i.e., 34-63%. Examination of the predicted secondary structure of alpha-momorcharin and trichosanthin indicates that these proteins have regions of high structural similarities and this may account for the common biological activities that they share, viz., abortificant, immunosuppressive, antitumor and inhibition of HIV-1.

XcmI as a universal restriction enzyme for single-stranded DNA.

Single-stranded DNA can be cleaved into defined fragments at any predetermined site by interaction with a specially designed oligodeoxyribonucleotide (oligo) adaptor and the class-IIN restriction endonuclease, XcmI. The oligo adaptor has the structure [sequence: see text]. Upon hybridization to the target DNA through the central 9-nucleotide region and with the addition of XcmI, the template DNA is specifically cleaved to near completion. Hairpin structures on the template close to the hybridization site reduce the efficacy of cleavage.

Cloning of trichosanthin cDNA and its expression in Escherichia coli.

Several cDNA clones coding for trichosanthin (TCS) have been isolated from a cDNA library prepared from the poly(A)+RNA of the root tuber of Trichosanthes kirilowii Maximowicz. The nucleotide sequence codes for a protein of 289 amino acids (aa) including a putative signal peptide of 23 aa and an extra 19 aa at the C terminus; the latter two have not been found in TCS obtained from the natural product [Collins et al., J. Biol. Chem. 265 (1990) 8665-8669]. Recombinant TCS (reTCS) was synthesized in Escherichia coli, in which the cDNA without the signal sequence was expressed under the control of the trc promoter; reTCS was detected by a rabbit anti-TCS antiserum.

Increasing the plasma half-life of trichosanthin by coupling to dextran.

Trichosanthin (TCS) is a plant protein which has a wide spectrum of pharmacological activities. It was demonstrated recently that this compound suppressed the replication of human immunodeficiency virus (HIV-1) in vitro. The mechanism of action is believed to be inhibition of protein synthesis. Trichosanthin is a low molecular weight protein which is expected to be easily filtered and eliminated through the kidney. To minimize renal loss, the molecular size of trichosanthin can be increased by coupling to dextran. The larger complex will not undergo glomerular filtration and therefore renal loss can be prevented. This study investigates the kidney's role in trichosanthin elimination and the beneficial effect afforded by coupling to dextran in prolonging plasma half-life. For this purpose, a radioimmunoassay has been developed to determine the concentration of TCS in plasma and urine. The sensitivity of this assay is in the nanogram range. Trichosanthin was coupled to dextran T40 by a dialdehyde method and successful coupling was confirmed by gel filtration chromatography. The complex retained specific binding to trichosanthin antibodies with decreased affinity which can be partially reversed after incubation with dextranase; an enzyme that digested dextran. The pharmacokinetics of intravenously administered trichosanthin (0.75 mg/kg) was compared between two groups of rats with normal and impaired renal function (bilateral renal arterial ligation). Rats with ligation showed a decrease in plasma clearance from 4780 +/- 570 to 220 +/- 20 microL/min and an increase in the mean residence time from 9 +/- 1 to 145 +/- 16 min. Despite the several-fold difference in these parameters, recovery of trichosanthin from normal rat urine was only 0.38 +/- 0.05%. This value can be increased by using higher injection doses. The data indicate that the kidney is an important organ for the elimination of trichosanthin. When the dextran-trichosanthin complex was injected into normal rats trichosanthin activity was not detected in the urine. All the pharmacokinetic parameters suggest that the dextran-trichosanthin complex stayed longer in the body and maintained a much higher plasma concentration than trichosanthin.

Lowering of trichosanthin immunogenicity by site-specific coupling to dextran.

Trichosanthin is a type I ribosome-inactivating protein possessing a broad spectrum of biological and pharmacological activities. Therapeutic use of this compound is hampered by its immunogenicity. It was shown earlier that coupling of dextran to trichosanthin can increase plasma half-life and reduce antigenicity. However, the site where dextran attaches to trichosanthin cannot be controlled; ideally, it should be at or near the antigenic determinant. The present study attempted to couple dextran to trichosanthin at a potential antigenic site. By site-directed mutagenesis, two sites, R29 and K173, were replaced by cysteine, and dextran was coupled to the newly created cysteine residues. The dextran-trichosanthin complex retained 50% of abortifacient activity and had a mean residence time in rats 27-fold longer than natural trichosanthin. Acute hypersensitivity reaction in guinea pigs was reduced greatly after coupling of K173C (a trichosanthin mutant with lysine-173 replaced by cysteine) to dextran. Compared with natural trichosanthin, dextran-K173C had a decrease in IgG and IgE response, whereas the coupling of R29C (a trichosanthin mutant with arginine-29 replaced by cysteine) to dextran did not show significant reduction of immunogenicity. This suggests that K173 but not R29 is located at or near an antigenic determinant. This study has demonstrated an alternative approach for mapping of antigenic determinants. The information obtained is also useful in producing an improved trichosanthin derivative for therapeutic use.

Molecular cloning and expression analysis of human glycogen synthase kinase-3 alpha promoter.

Human glycogen synthase kinase-3 alpha (GSK-3 alpha) is a serine/threonine kinase that phosphorylates a variety of cytoplasmic and nuclear proteins. It also phosphorylates components of the neuronal cytoskeleton including tau and neurofilament heavy chain. Hyperphosphorylated tau is found in neurofibrillary tangles, a hallmark of Alzheimer's disease and aberrant phosphorylation of neurofilament heavy chain is observed in motor neuron disease. Alterations in GSK-3 alpha activity may therefore contribute to the disease process in these disorders. As a first step to understand the transcriptional regulation of GSK-3 alpha, a 2-kb (p-1751/+243) DNA fragment upstream of the GSK-3 alpha initiation codon was obtained from a YAC clone and characterised. Using primer extension assays, a putative transcriptional start site was located to a G nucleotide 244 bp upstream of the ATG codon. Several transcription factor-binding sites were identified on the promoter region, but no TATA-like element was located close to the start site. Deletion mutants of the 2-kb DNA fragment were generated and fused to a promoterless chloramphenicol acetyltransferase (CAT) gene. Transfection study in a neuroblastoma cell line revealed the 1-kb (p-719/+243) fragment carried strong promoter activity, while the 2-kb construct that contains an Alu-like sequence was only 50% active.

Restriction endonucleases in clinical isolates of Shigella spp.

Thirteen restriction endonuclease-containing strains were isolated from a collection of 186 clinical isolates of Shigella spp. Among these, eight and five isolates carried isoschizomers of EcoRII and NciI, respectively. The former restriction-modification (R-M) system was homologous to that of EcoRII and was located on plasmids with sizes of 46.6 or 55.6 kb. Isolates producing NciI isoschizomers contained a 5.7-kb non-transferable plasmid. Together with antimicrobial susceptibility tests and plasmid profile studies, it is concluded that these two R-M systems are not widely spread but confined to strains with similar antibiotic resistance and plasmid profile.

Postoperative stomach and duodenum.

A hypotonic biphasic contrast study proves or excludes ulceration and neoplasm in most instances. As in nonoperated patients, an initial radiologic examination may therefore serve as a screening method to determine whether endoscopy is indicated. After surgery artifacts may occur, which in some cases cannot be differentiated from malignant tumors or ulcer craters on a radiologic basis alone, although postoperative baseline studies may be helpful. In operated patients endoscopy is needed in a higher percentage than in nonoperated patients. Furthermore, in our experience endoscopy has proved to be superior to radiology in detecting small jejunal ulcers after a Billroth II resection. The possibility of recurrent carcinoma must be considered even after a short interval following gastric carcinoma surgery; however, if surgery was undertaken for a benign lesion, a higher rate of malignancy (primary gastric stump carcinoma) is not to be expected before a postoperative interval of at least 5 years.

Importance of the Glu 160 and Glu 189 residues to the various biological activities of the ribosome inactivating protein trichosanthin.

Site-directed mutagenesis of trichosanthin (TCS), a ribosome inactivating protein with a broad spectrum of biological activities, was carried out to ascertain the importance of the Glu 160 and Glu 189 residues to the protein synthesis-inhibitory, antiproliferative, immunosuppressive and embryotoxic activities of TCS. Replacement of Glu 160 with alanine and with aspartate produced muteins, designated [E160A] and [E160D] respectively, with considerably attenuated protein synthesis-inhibitory, antiproliferative, immunosuppressive and embryotoxic activities, indicating that Glu 160 in TCS plays a role in its biological activity. [E160A] was, however, more potent than [E160D] because in the former mutein, Glu 189 constitutes a back-up of the carboxylate group but in the latter mutein, the negative charge from Asp is at a suboptimal position. The mutein [E160A, E189A] formed by mutation of both Glu 160 and Glu 189 retained considerable embryotoxic activity, suggesting that other amino acids in the active site were able to partially replace the role of Glu 160 and Glu 189. The TCS muteins also exhibit higher toxicity toward cultured embryos than cultured cells (spleen cells and tumor cells).

Reducing the immunogenicity and improving the in vivo activity of trichosanthin by site-directed pegylation.

PEG modification (PEGylation) has been shown to reduce immunogenicity and prolong circulating half-life of proteins. In the present study, site-directed PEGylation was used to reduce immunogenicity and prolong plasma half-life of trichosanthin (TCS). Four TCS mutants, i.e. S7C, Q219C, K173C and [K173C,Q219C] (KQ), were constructed by site-directed mutagenesis. PEG modifications were done by reacting PEG5k-maleimide or PEG20k-maleimide reagent with the newly introduced cysteine residue of the mutants. The plasma clearance rate of PEGylated TCS mutants decreased up to 100-fold and the decrease was inversely proportional to the effective molecular size. The in vitro activities such as ribosome-inactivating activity and cytotoxicity were also decreased. However, the in vivo abortifacient activity was, slightly decreased, unchanged, or even enhanced in some preparations. PEG5k modification had little effect on immunogenicity. However, PEG20k modification significantly reduced immunogenicity. All PEG20k modified TCS mutants induced lower level IgG and IgE antibodies. In particular, PEG20k-KQ and PEG20k-K173C induced weaker systemic anaphylaxis reaction in guinea pigs. In conclusion, the present results suggest that PEG20k is better than PEG5k for reducing immunogenicity and prolonging plasma half-life. The conjugate can become a better therapeutic agent.

Minireview: trichosanthin--a protein with multiple pharmacological properties.

Trichosanthin (TCS), a protein isolated from the root tuber of the Chinese medicinal herb Trichosanthes kirilowii Maxim., is used to induce abortion in China. It also possesses immunomodulatory, anti-tumor and anti-human immunodeficiency virus properties. TCS is a member of the family of ribosome-inactivating proteins and inactivates eukaryotic ribosomes via its N-glycosidase activity. The gene encoding TCS has been cloned and over-expressed and the crystal structure of this protein resolved to 1.73A. In this review, the various pharmacological properties of TCS are discussed and assessed.

Site-directed polyethylene glycol modification of trichosanthin: effects on its biological activities, pharmacokinetics, and antigenicity.

Trichosanthin (TCS), a type I ribosome-inactivating protein (RIP), was modified with polyethylene glycol (PEG) in order to reduce its antigenicity and prolong its half-life. Computer modeling identified three potential antigenic sites namely Q219, K173 and S7. By site-directed mutagenesis, these sites were changed into cysteine through which PEG can be covalently attached. The resulting TCS had a PEG coupled directly above one of its potential antigenic determinants, hence masking the antigenic region and prevent binding of antibodies specific to this site. In general, mutation did not bring about significant changes in ribosome-inactivating activity, cytotoxicity, and abortifacient activity of TCS. However, the in vitro activities of PEG modified (PEGylated) TCS muteins were 3-20 folds lower and the in vivo activity 50% less than that of nTCS. Pharmacokinetics study indicated that all three PEGylated TCS muteins showed 6-fold increase in mean residence time as compared to unmodified muteins. The binding affinity of an IgE monoclonal antibody (TE1) to TCS was greatly reduced after PEG modification (PEGylation) at position Q219, suggesting that TE1 recognized an epitope very near to residue Q219. PEGylated TCS muteins induced similar IgG response but 4-16 fold lower IgE response in mice compared with nTCS.

The antigenic sites of trichosanthin, a ribosome-inactivating protein with multiple pharmacological properties.

Trichosanthin (TCS) fragments were produced by Tn1000 deletion mutagenesis and by cyanogen bromide (CNBr) cleavage and their immunoreactivity was examined by incubating with various antibodies. Twelve C-terminally truncated TCS variants were successfully synthesized under the control of a T7 RNA driven promoter. The smallest antigenic fragment mapped corresponded to the N-terminal 20 amino acids (aa). Six CNBr fragments of TCS were created and identified by electrospray mass spectrometry and N-terminal sequencing. Three antigenic fragments corresponding to aa 1-72, 101-152 and 153-246, respectively were mapped. Fragments corresponding to aa 1-72 and 153-246 were immunoreactive to the same monoclonal antibody showing they are components of a discontinuous epitope. On the other hand, the fragment containing aa 73-100 was not detected by any of the antibodies used.

Absence of Epstein-Barr virus (EBV) in a gastrointestinal stromal cell tumour (GIST) in an adult human immunodeficiency virus-seropositive patient with past EBV infection.

Gastrointestinal stromal cell tumours (GIST) of the small intestine are rare malignancies. Recently, an association of Epstein-Barr virus (EBV) with malignant stromal cell tumour in young people with AIDS and past EBV infection has been described. We describe a 33-year-old heterosexual male with asymptomatic human immunodeficiency virus (HIV) infection who had had an EBV infection in the past and who presented with an EBV-negative GIST. The association between EBV and malignant stromal cell tumours in young people with AIDS could not be reconfirmed in our adult patient. The relationship between EBV and malignant stromal cell tumours in AIDS patients and the possible pathogenetic role of EBV remains to be established, at least in adults.

Presence of strA-strB gene within a streptomycin-resistance operon in a clinical isolate of Shigella flexneri.

Three aminoglycoside-modifying enzymes (AMEs) were produced by a clinical isolate of Shigella flexneri which was resistant to gentamicin, tobramycin, netilmicin, kanamycin, sisomicin and streptomycin: acetyltransferase (AAC) (3)-II-type, and phosphotransferase (APH) (3")- and (6)-type enzymes. The aminoglycoside-resistance genes were located on a 75-Kb plasmid. Two genes, strA-HK and strB-HK, in a transcriptional unit were found to code for streptomycin-resistance. The genetic organization and sequence of this transcriptional unit were identical to those of strA and strB in plasmid RSF1010. strA-HK and strB-HK when expressed separately produced functional enzymes. Our substrate profile study on the crude extracts of StrA-HK and StrB-HK proteins confirmed that StrA-HK was an APH(3")-type and showed that StrB-HK was a member of the APH(6) family.

Physiotherapist-led programmes and interventions for rehabilitation of anterior cruciate ligament, medial collateral ligament and meniscal injuries of the knee in adults.

BACKGROUND: Soft-tissue injuries of the knee, mainly involving the anterior cruciate ligament (ACL), the medial collateral ligament (MCL) and menisci, are common and their rehabilitation after non-surgical or surgical treatment often involves intensive and prolonged physiotherapy. OBJECTIVES: To examine the evidence for effectiveness of various physiotherapist-led (or 'directed') rehabilitation programmes, and of various interventions used within these programmes, for rehabilitation of acute or chronic ACL, MCL or meniscal injuries of the knee in adults. SEARCH STRATEGY: We searched the Cochrane Musculoskeletal Injuries Group's specialised register (to June 2001), MEDLINE (from 1966 to August 1999), EMBASE (from 1980 to February 1997), CINAHL (1982 to April 1999), CURRENT CONTENTS (up to March 1999) and reference lists of relevant articles, and consulted colleagues. Date of the most recent search: June 2001. SELECTION CRITERIA: Randomised or quasi-randomised clinical trials evaluating physiotherapist-led rehabilitation programmes, or components of rehabilitation programmes, for the treatment or post-surgical rehabilitation of ACL, MCL or knee meniscal injuries. Excluded were trials investigating electrical stimulation, or various interventions such as cryotherapy, immobilisation braces and continuous passive motion when used in initial or early treatment. Laboratory based trials reporting intermediate outcomes were also excluded. DATA COLLECTION AND ANALYSIS: All trials, judged as fitting the selection criteria by two reviewers, were independently assessed by two reviewers for methodological quality by use of an 11 item checklist. Data were independently extracted by two reviewers. Any disagreement was resolved by discussion. Although quantitative data from most trials are presented, using relative risks or mean differences together with 95 per cent confidence intervals, trial heterogeneity and lack of outcome data prevented meaningful pooling of results from comparable trials. MAIN RESULTS: Thirty-one trials, involving 1545 mainly young and male patients, met the inclusion criteria of the review. Methodological quality was highly variable: allocation concealment and / or assessor blinding were rare, and assessment of outcome was often incomplete and short-term. ACL injury and /or deficiency was the main focus of 18 trials, MCL injury of two trials, meniscal injury of nine trials and a mixture of soft-tissue injuries in the other two trials. The trial comparisons fell into five main categories: rehabilitation programme versus control (6 trials); one rehabilitation programme versus another (6 trials); different timing of rehabilitation (4 trials); one component of a programme versus another (6 trials); supplementary interventions to a programme versus none (9 trials). No trial provided sufficient evidence to establish the relative effectiveness of the intervention(s) under investigation. REVIEWER'S CONCLUSIONS: The available evidence for physiotherapist-led rehabilitation of ACL, MCL and meniscal injuries is wide ranging in terms of scope but insufficient to establish the relative effectiveness of the various approaches and methods in current use. There is a need for further research involving good quality, large scale randomised trials with sufficiently long follow-up to fully assess knee function and recovery.

Authentication of Panax ginseng and Panax quinquefolius using amplified fragment length polymorphism (AFLP) and directed amplification of minisatellite region DNA (DAMD).

AFLP profiles characteristic to Panax ginseng and Panax quinquefolius were generated using primers E-AGG/M-CAA. P. ginseng samples from different farms in China and Korea are homogeneous genetically [similarity index (SI) = 0.88-0.99], whereas samples of P. quinquefolius from different sources are much more heterogeneous (SI = 0.64-0.96). Detailed analysis of one of the polymorphic bands in P. ginseng led to the identification of a minisatellite Pg2, which contains eight repeats of 5'-AGGACTCATCACATTGTTACTC. The minisatellite DNA was consequently used in directed amplification minisatellite region DNA analysis to authenticate the two ginsengs.