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.

Recent progress in medicinal investigations on trichosanthin and other ribosome inactivating proteins from the plant genus Trichosanthes.

Ribosome inactivating proteins (RIPs) are toxic RNA N-glycosidases that cleave an adenine-ribose glycosidic bond at position adenine(4324) with the conserved ricin/α-sarcin loop in the eukaryotic 28S ribosomal RNA. RIPs have captured the attention of botanists, biochemists, and drug discoverers, due to their diverse potent defensive activities, and inter alia, their antitumor and anti-HIV activities. Out of the 145 families of plants, Trichosanthes ranks among the top 5 genera with a good potential of use for discovery of anticancer drugs. Trichosanthin (TCS) is a famous type I RIP purified from T. kirilowii that has been known for around 30 years. Based on the results of voluminous in vitro and in vivo investigations, TCS is considered a good candidate for the treatment of HIV/AIDS and neoplasms. Here we integrate recent progress of the research on the different medicinal activities of TCS. In addition to TCS, other promising RIPs from the same species (such as TAP29 and trichoanguin), and from the same genus Trichosanthes are included. This review presents a brief panorama of the studies on Trichosanthes RIPs. Regarding the debilitating nature of AIDS and different tumors, further understanding of these multifunctional proteins is worthwhile since it may help to open a novel therapeutic window for these stubborn diseases.

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.

BRE is an antiapoptotic protein in vivo and overexpressed in human hepatocellular carcinoma.

BRE binds to the cytoplasmic domains of tumor necrosis factor receptor-1 and Fas, and in cell lines can attenuate death receptor-initiated apoptosis by inhibiting t-BID-induced activation of the mitochondrial apoptotic pathway. Overexpression of BRE by transfection can also attenuate intrinsic apoptosis and promote growth of the transfected Lewis lung carcinoma line in mice. There is, however, a complete lack of in vivo data about the protein. Here, we report that by using our BRE-specific monoclonal antibody on the immunohistochemistry of 123 specimens of human hepatocellular carcinoma (HCC), significant differences in BRE expression levels between the paired tumoral and non-tumoral regions (P<2.2e-16) were found. Marked overexpression of BRE was detected in majority of the tumors, whereas most non-tumoral regions expressed the same low level of the protein as in normal livers. To investigate whether BRE overexpression could promote cell survival in vivo, liver-specific transgenic BRE mice were generated and found to be significantly resistant to Fas-mediated lethal hepatic apoptosis. The transgenic model also revealed post-transcriptional regulation of Bre level in the liver, which was not observed in HCC and non-HCC cell lines. Indeed, all cell lines analysed express high levels of BRE. In conclusion, BRE is antiapoptotic in vivo, and may promote tumorigenesis when overexpressed.

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.

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.

Application of sequence characterized amplified region (SCAR) analysis to authenticate Panax species and their adulterants.

A 420-bp RAPD fragment from Panax quinquefolius was converted to a sequence characterized amplified region (SCAR) marker. The main difference between the SCAR of P. quinquefolius and its homolog in P. ginseng is the presence of a 25 bp insertion in the latter. Primers derived from this sequence were successfully used to authenticate six Panax species and two common adulterants.

Direct amplification of length polymorphism analysis differentiates Panax ginseng from P. quinquefolius.

The method of direct amplification of length polymorphism (DALP) was applied to authenticate Panax ginseng and P. quinquefolius. A 636-bp DALP fragment was present in all P. ginseng but absent in all the P. quinquefolius cultivars examined. We have shown that the use of DALP and conversion of specific polymorphic band to sequence-tagged site (STS) for quick authentication may be applied to authenticate related medicinal materials.

Authentication of medicinal Dendrobium species by the internal transcribed spacer of ribosomal DNA.

Herba Dendrobii (Shihu) is a commonly used Chinese medicine derived from the stem of several orchid species belonging to the genus Dendrobium. It is rather expensive and adulteration is frequent. Proper authentication of the medicinal species is necessary to protect consumers and support conservation measures. DNA sequences of the internal transcribed spacer 2 (ITS 2) of 16 Dendrobium species were shown to be significantly different from one another by an average of 12.4% and from non-orchids and Pholidota (an adulterant of Shihu) by 29.8% and 18.8%, respectively. The intra-specific variation among the Dendrobium species studied was only about 1%. Therefore, ITS 2 regions could be adopted as a molecular marker for differentiating medicinal Dendrobium species from one another and also from non-orchids and adulterants.

Trichosanthin interacts with acidic ribosomal proteins P0 and P1 and mitotic checkpoint protein MAD2B.

Trichosanthin is a ribosome-inactivating protein with multiple pharmacological properties. By a yeast two-hybrid system, ribosomal phosphoproteins P0 and P1 and a putative mitotic checkpoint protein, MAD2B, were found to interact with an active-site mutated trichosanthin (TCS). The interactions were verified by an in vitro binding assay of recombinant wild-type TCS and target proteins. The interaction domain of P0 was mapped to amino acids 220-273, which had been previously reported to be involved in the interaction with P1 and P2 in yeast. Consistent with our previous finding that the last seven residues of TCS are not essential for an active conformation, the same deletion did not affect the interaction with P0. Our present study suggests that TCS may disrupt the binding of elongation factors to the P-complex, in addition to the well-known N-glycosidase activity for ribosome inactivation.

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.

Engineering of a mini-trichosanthin that has lower antigenicity by deleting its C-terminal amino acid residues.

Trichosanthin is a ribosome-inactivating protein that possesses antitumor and antiviral activities. Clinical trials of trichosanthin on AIDS patients, however, elicit anaphylactic reactions. To reduce the antigenicity of trichosanthin as a drug while preserving its biological activity, the C-terminal domain (residues 203 to 247), which contains a putative antigenic site, was systemically deleted. We have found that the minimum length of trichosanthin that can fold into an active conformation is residue 1 to 240. The mini-trichosanthin (C7) generated by deleting the last seven C-terminal amino acid residues has 2.7-fold decrease in antigenicity, 10-fold reduction in in vitro ribosome-inactivation activity, and in vivo cytotoxicity toward K562 cells, and 2-fold reduction in abortificient activity. Structural analyses of C7 indicate decrease in the helix content, increased exposure of Trp192, and lower thermodynamic stability. The deletion of the C-terminal residues (Leu241 to Ala247) probably perturbs local structure of the C-terminal antigenic epitope that results in the decrease in antigenicity and activities of C7.

Trichosanthin interacts with and enters cells via LDL receptor family members.

The type-I ribosome-inactivating protein trichosanthin displays selective cytotoxicity, suggesting specific mechanisms for entry into cells. Here we show that trichosanthin binds specifically to the endocytic receptors LRP and megalin, and that binding as well as uptake into cells is inhibited by the receptor-associated protein (RAP). The results suggest that the known abortifacient and renotoxic actions of trichosanthin are caused by LRP-mediated uptake in trophoblasts and megalin-mediated uptake in proximal tubule epithelial cells, respectively.

Differentiation of medicinal Codonopsis species from adulterants by polymerase chain reaction-restriction fragment length polymorphism.

DNA sequence analysis of rDNA internal transcribed spacer (ITS) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) were exploited for their applications in differentiating medicinal species Codonopsis pilosula, C. tangshen, C. modesta, and C. nervosa var. macrantha, from two related adulterants Campanumoea javania and Platycodon grandiflorus. The data demonstrated that the rDNA ITSI and ITSII sequences of the four Codonopsis are highly homologous but not identical, and are significantly different from those of the two adulterants. The sequence difference allows effective and reliable differentiation of Codonopsis from the adulterants by PCR-RFLP.

Molecular cloning and characterization of the human glycogen synthase kinase-3beta promoter.

Glycogen synthase kinase 3beta (GSK-3beta) is a proline-directed kinase that forms part of the wingless signaling pathway. Recent studies have shown that GSK-3beta phosphorylates the microtubule-associated protein tau in vitro and in cell culture. Tau is the principal component of the paired helical filaments (PHFs) found in the brains of patients with Alzheimer disease, and PHF-tau is hyperphosphorylated. GSK-3beta is therefore one of the candidate kinases for phosphorylating tau in Alzheimer disease. GSK-3beta activity is negatively regulated by phosphorylation on serine 9 and positively regulated by phosphorylation on tyrosine 216. However, since overexpression of GSK-3beta by transfection leads to increased activity in the absence of any stimuli, GSK-3beta activity may also be regulated at the transcriptional level. Indeed, increased GSK-3beta protein levels are found in Alzheimer disease brains, and GSK-3beta is found associated with PHFs in Alzheimer disease. To understand how GSK-3beta activity may be regulated at the transcriptional level, we have isolated the human GSK-3beta promoter. The GSK-3beta promoter does not contain a conventional TATA box although several other transcription factor binding sites were identified. A putative transcription start site was mapped by 5' RACE. Transfection of various GSK-3beta promoter CAT reporter genes into both COS-7 cells and SHSY5Y neuronal cells revealed that the GSK-3beta promoter is more active in neuronal cells. Such transfection studies involving promoter deletion mutants revealed that a negative transcriptional response element may be present at position -1421 to -1363 and an activator sequence at position -427 to -384. CP2 binding sites were also present within the promoter. CP2 has recently been shown to interact with the Alzheimer disease amyloid precursor protein binding protein Fe65. The significance of these results with respect to Alzheimer disease pathogenesis are discussed.

Expression analysis of glycogen synthase kinase-3 in human tissues.

Human glycogen synthase kinase-3 (GSK-3) is a multisubstrate, proline-directed kinase that phosphorylates tau, beta-amyloid and neurofilaments. In this study, the expression levels of the two GSK-3 isoforms, alpha and beta, RNA and proteins in different human tissues were examined. Northern analysis demonstrated that GSK-3alpha is encoded by a 2.6-kb mRNA and GSK-3beta by 8.3- and 2.8-kb mRNAs. The two GSK-3beta mRNA species were variably expressed in different tissues. Northern and quantitative polymerase chain reaction demonstrated that both GSK-3alpha and GSK-3beta mRNA were prominently expressed in testis, thymus, prostate and ovary but were low in adult lung and kidney. Western blot analysis showed that the 51-kDa GSK-3alpha protein was highly expressed in lung, ovary, kidney and testis, whereas the 46-kDa GSK-3beta protein was highly expressed in lung, kidney and brain. The differential expression of GSK-3alpha and GSK-3beta mRNA and proteins and the lack of relationship between transcription and translation in some tissues indicate that GSK-3alpha and GSK-3beta are subject to different means of regulation.

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.

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.

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.