Biomedical subjects

P C Shaw

Publications and source records attributed to P C Shaw.

At least 37 records · Page 2Linked to original sources

PubMed1997

Structure/function relationship study of Tyr14 and Arg22 in trichosanthin, a ribosome-inactivating protein.

Amino acids Tyr14 and Arg22 in trichosanthin are residues on helix A1 close to the active-site cleft. They are invariant in various type-I and type-II ribosome-inactivating proteins. In this study, Tyr14 was changed to Phe and Arg22 to Lys and Leu. Modified proteins were purified, and activities compared by assaying their median inhibitory concentration (ID50) on a rabbit-reticulocyte-lysate protein-synthesis system. While the ID50 of wild-type trichosanthin was 0.02 nM, those for [Phe14], [Lys22], [Leu22] and [Phe14, Leu22]trichosanthin were 0.10, 0.03, 0.25 and 0.15 nM, respectively. Therefore, compared with Tyr14, Arg22 appears to play a more important role in trichosanthin. Structural studies on [Leu22]trichosanthin showed that two water molecules occupy the space left by the side chain of Arg22, and hydrogen bonds exist between these water molecules and nearby residues to retain the conformation. The use of intermolecular rather than intramolecular hydrogen bonds may have an adverse effect on stability or folding of the protein and results in a mild decrease in activity.

Abortifacient Agents, Nonsteroidal↗

PubMed1997

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.

Animals↗

PubMed1997

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.

Adult↗

PubMed1997

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.

Anti-Bacterial Agents↗

PubMed1996

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).

Animals↗

PubMed1996

Overproduction, purification and characterization of M.EcoHK31I, a bacterial methyltransferase with two polypeptides.

The two overlapping genes coding for EcoHK31I methyltransferase have previously been cloned, sequenced and expressed [Lee, Kam and Shaw (1995) Nucleic Acids Res. 23, 103-108]. Here we describe protocols developed to purify polypeptides alpha and beta together or separately, to apparent homogeneity by various chromatographic media. M.EcoHK31I is a heterodimer with a native molecular mass of 61 kDa. Its specific activity towards non-methylated lambda DNA was 3.0 x 10(5) units per mg of protein. The respective denatured molecular masses of polypeptides alpha and beta were 38 and 23 kDa, and their pI values were 8.7 and 6.8. Initial rate kinetic parameters of the native enzyme were 2.0 nM, 0.58 microM and 3 min-1 for KmDNA, KmAdoMet and kcat. respectively, where AdoMet stands for S-adenosyl-L-methionine. Fully active enzyme was reconstituted by co-purifying the two separately synthesized polypeptides, and activity assays confirmed our previous finding that two polypeptides were needed to methylate substrate DNA.

Amino Acid Sequence↗

PubMed1996

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.

Drug Resistance, Microbial↗

PubMed1996

Authentication of the Chinese drug "ku-di-dan" (herba elephantopi) and its substitutes using random-primed polymerase chain reaction (PCR).

DNA fingerprinting and polymorphism among the Chinese drug "Ku-Di-Dan" and its substitutes were demonstrated with arbitrarily primed polymerase chain reaction (AP-PCR) and random amplified polymorphic DNA (RAPD). Distinctive, reproducible genomic fingerprints from DNA of the Chinese drug Ku-Di-Dan and 9 species of Compositae were generated with six long (18-24 mer) and one short (10 mer) random-chosen primers with PCR. Ku-Di-Dan samples can be distinguished according to the banding patterns of their amplified DNA on agarose gels. Results showed that AP-PCR and RAPD fingerprints of the commercial samples of Ku-Di-Dan retailed in Fujian, Taiwan, Hong Kong and Macau markets are identical with that of Elephantopus scaber L. The relatedness of Ku-Di-Dan among 9 plants may also be estimated by the Similarity Indexes values of the genomic DNA fingerprints.

Asteraceae↗

PubMed1995

A bacterial methyltransferase M.EcoHK311 requires two proteins for in vitro methylation.

The genes encoding EcoHK311 restriction-modification (R-M) system were isolated from a clinically-isolated Escherichia coli strain HK31. The entire R-M system of EcoHK311 is located in a 2.1 kb fragment. R.EcoHK311 is an isoschizomer of Eael which recognizes and cleaves Y decreases GGCCR. M.EcoHK31l consists of two polypeptides alpha and beta with sizes 309 and 176 aa, respectively. Polypeptide beta is encoded within aa, alternative reading frame of polypeptide alpha. All the conserved motifs in mC5-MTases can be found in polypeptide alpha except motif IX which is present in polypeptide beta. Polypeptides alpha and beta were separately synthesized in a T7 promoter controlled over-expression system and in vitro methylation occurred only when the two extracts were mixed and thus confirms that two polypeptides are required for methylation.

Amino Acid Sequence↗

PubMed1995

Mouse embryonic development and tumor cell growth under the influence of recombinant trichosanthin (a ribosome inactivating protein) and its muteins.

Trichosanthin, a ribosome inactivating protein, is known to possess embryotoxic and antiproliferative activities. In the present study recombinant trichosanthin and its muteins produced by site-directed mutagenesis were tested for these activities using whole embryo culture and cell culture. The results revealed that when Glu 160 in trichosanthin was mutated to alanine or aspartate, the two aforementioned activities underwent a substantial decrease. However, the former mutein had a higher potency than the latter mutein. When both Glu 160 and Glu 189 were mutated, the resultant mutant possessed very low antiproliferative and yet considerable embryotoxic activity, indicating the contribution from other amino acids in stabilizing the transition state complex of trichosanthin.

Animals↗

PubMed1995

Authentication of Panax species and their adulterants by random-primed polymerase chain reaction.

DNA from dried or fresh roots of three medicinal Panax species and their adulterants were amplified by AP-PCR or RAPD. Fingerprints for P. ginseng or P. quinquefolius were found to be consistent irrespective of plant source or age. On the other hand, very different fingerprints were obtained from adulterants. Hence the PCR approach may be used as a means to authenticate the concerned Panax species. Analysis of the degree of similarity of the fingerprints confirmed that P. ginseng is more closely related to P. quinquefolius than to P. notoginseng.

Base Sequence↗

PubMed1994

Structure/function relationship study of Gln156, Glu160 and Glu189 in the active site of trichosanthin.

Trichosanthin is a protein used medicinally in China for abortifacient purposes. It is also an RNA N-glycosidase which inactivates eukaryotic ribosomes by removing adenine4324 from 28S rRNA. Site-directed mutagenesis was performed to probe the role of Gln156, Glu160 and Glu189 in the active site of trichosanthin. The purified altered proteins were assayed for their potency in inhibiting in vitro protein synthesis. The data indicate Glu160 is involved in the catalytic reaction. Kinetics studies suggest the carboxylate group of Glu160 serves to stabilize the transition-state complex. Similar to ricin A, the variant [E160A]trichosanthin is more potent than [E160D]trichosanthin. This is because Glu189 serves as a back-up of the carboxylate group in case Glu160 is mutated to alanine. However, removal of Glu189 in the presence of Glu160 does not affect the ID50 value drastically. An activity of 1800-fold less than that of the wild-type protein was found when both Glu160 and Glu189 were changed to alanine, indicating that some other residues in the active site are also taken part in the lowering of energy barrier for the catalytic reaction. Although Gln156 is highly conserved in related proteins, its mutation to alanine only slightly decreases the activity, showing that this residue does not participate directly in catalysis.

Amino Acid Sequence↗

PubMed1994

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.

Abortifacient Agents↗

PubMed1994

Pharmacognostical identification of American and Oriental ginseng roots by genomic fingerprinting using arbitrarily primed polymerase chain reaction (AP-PCR).

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Base Sequence↗

PubMed1994

Case report: aneurysm of an aberrant right subclavian artery diagnosed with MR imaging.

An aberrant right subclavian artery (ARSCA), also known as arteria lusoria, is the most common congenital anomaly of the aortic arch, with a reported prevalence ranging from 0.4 to 2%. The ARSCA arises as the last branch of the aortic arch and crosses the mediastinum from left to right indenting the oesophagus posteriorly. Aneurysms of this aberrant vessel, whether or not arising from a Kommerell's diverticulum, are rare. A 79-year-old woman is presented, in whom a partly thrombosed aneurysm of an ARSCA was diagnosed with magnetic resonance (MR) imaging.

Aged↗

PubMed1994

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.

Anastomosis, Surgical↗

PubMed1993

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.

Base Sequence↗

PubMed1993

The role of early baseline computed tomography in the interpretation of morphological changes after kidney-pancreas transplantation.

In a prospective study, 17 early baseline computed tomography (CT) scans were obtained 2 or 3 days after simultaneous kidney-pancreas transplantation. Morphological changes and their relevance to the early detection of graft rejection and complications were evaluated. The pancreatic grafts were enlarged and showed signs of mild pancreatitis. Serial scans obtained during the first renal graft rejection episode were compared with the baseline CT scans (n = 7). They showed a significant increase in pancreatic graft size in the case of biopsy-proven severe renal graft rejection (P = 0.008). Normally functioning pancreatic allografts showed a 15%-40% decrease in size 1-6 months after transplantation. We conclude that the morphological changes observed early after transplantation are compatible with mild pancreatitis, which may contribute to the development of pancreatic graft thrombosis. There is an increase in the number of morphological changes during severe rejection, yet enlarged pancreatic grafts appear to recover from transplantation-related damage and regain their normal size without signs of atrophy.

Adolescent↗