Biomedical subjects

S R Lin

Publications and source records attributed to S R Lin.

At least 37 records · Page 2Linked to original sources

PubMed1995

Characterization of epitopes in native and unfolded cobrotoxin: evidence of an immunodominant C-terminal region related to the production of precipitating and non-precipitating antibodies against cobrotoxin.

Rabbits hyperimmunized with cobrotoxin from Taiwan cobra venom produced non-precipitating as well as precipitating antibodies. Both antibody preparations exhibited higher affinity for native cobrotoxin than for reduced and S-carboxymethylated (RCM) cobrotoxin. This indicated that the epitope structures in cobrotoxin are mostly conformation-dependent. In order to identify the conformational epitopes, native cobrotoxin was hydrolyzed with acid protease A, and 12 peptides were obtained on HPLC. Three peptide fragments, AP-10, AP-11, and AP-12, showed pronounced antigenicities toward precipitating as well as non-precipitating antibodies. AP-10, AP-11, and AP-12 contained a common segment in the C-terminal region of cobrotoxin, residues 43 to 62, with intact disulfide linkages. Complete removal of the C-terminal antibodies from antisera and precipitating antibodies on a C-terminal segment-Sepharose affinity column resulted in the loss of their precipitability with cobrotoxin, whilst restoration of precipitability was observed on the addition of the C-terminal antibodies to the C-terminal antibody-depleted antisera and precipitating antibodies. Studies on the antigenic structures of RCM-cobrotoxin revealed that RCM-cobrotoxin contains an immunodominant epitope at positions 22-38. The N-terminal and C-terminal regions of RCM-cobrotoxin encompass other epitopes which exhibit low reactivities toward anti-RCM-cobrotoxin antibodies. However, no precipitated antigen-antibody complexes were observed with the mixture of anti-RCM-cobrotoxin antibodies and RCM-cobrotoxin. These results suggest that the inherently different immunogenicities with different segments might affect the precipitabilities of the resulting antibodies, and that the notable immunogenecity of the C-terminal region is related to the production of precipitating and non-precipitating antibodies against cobrotoxin.

Amino Acid Sequence↗

PubMed1995

Genetic diagnosis of haemophilia A of Chinese origin.

Genetic diagnosis of haemophilia A has been studied in two aspects. One is to directly identify the mutations in the factor VIII genes of the affected probands, and the other is to examine the usefulness of several intragenic factor VIII markers for gene tracking. Direct mutational analysis by PCR-SSCP (polymerase chain reaction--single-strand conformation polymorphism) has been accomplished previously in 87 haemophilia A patients, accounting for nearly 10% of cases in Taiwan. Of the 87 cases, 46% were with point mutations, short deletions or insertions, and most of the remaining were with gene inversion readily identified by Southern blotting. Further examination of 112 patients has estimated a 33% incidence for gene inversion in all the patients with haemophilia A, or 37% in severe cases. Since the direct mutational detection described above cannot be used in all Chinese families with haemophilia A, genetic markers were also investigated. The two CA repeat markers located at intron 13 (CA-13) and intron 22 (CA-22), respectively, were amplified and analysed simultaneously. Seven different alleles with 18-24 CAs have been identified for CA-13. Alleles of 20 and 21 CAs are the most common and their population frequency was 0.68 and 0.24, respectively. The CA-22 marker contained a repetition of (GT)n(AG)n as was identified in the white European but not in the Canadian population. Alleles with 25 and 26 GT/AGs account for 18% and 75% of this group of samples, respectively. The expected rate of heterozygosity for either CA markers was 68%, although a value of 57% was observed by haplotype analysis, indicating an association of the two repeat markers. Nevertheless, the study of 62 females showed that with the combined use of CA-13 and CA-22 with BclI, approximately 71% would be informative for these markers. This number may increase to 81% if XbaI polymorphism is added. We propose that a better genetic diagnosis procedure for Chinese individuals would be first to look for the inversion mutation, secondly for one of the intragenic markers, and then at the PCR-SSCP analysis.

Blotting, Southern↗

PubMed1995

Decrease of renal endothelin 1 content and gene expression in diabetic rats with moderate hyperglycemia.

To investigate the intrarenal endothelin 1 (ET-1) synthesis in streptozocin (STZ) diabetic rats with moderate hyperglycemia, we measured plasma ET-1, renal ET-1 mRNA, and renal tissue ET-1 levels. The renal ET-1 mRNA expression progressively decreased from the 2nd to the 6th week after induction of diabetes by STZ. The renal ET-1 mRNA expression and the renal tissue ET-1 content were significantly reduced in 8 diabetic rats with a mean blood glucose level of 21.0 +/- 0.4 mM as compared with 7 normal rats sacrificed at the 6th week after STZ or citric buffer injection. The reduction of renal ET-1 and mRNA levels was ameliorated in 9 diabetic rats with a mean blood glucose level of 6.9 +/- 0.7 mM after strict glycemic control by insulin treatment. Kidney weight and glomerular filtration rate in moderately hyperglycemic rats were significantly increased as compared with normal rats at the 6th week after STZ injection. The mean plasma ET-1 levels in moderately hyperglycemic diabetic rats were not different from those of the other two groups. This study demonstrates that moderate hyperglycemia in diabetic rats is associated with a reduction in renal ET-1 synthesis. Whether decreased renal ET-1 synthesis is an adaptive phenomenon of a renal hemodynamic change during the early stage of diabetes is worthy of further investigation.

Animals↗

PubMed1995

Evaluation of the severity of Helicobacter pylori infection with urease test: its correlation with histopathology and bacterial density.

In 69 patients, the severity of Helicobactor pylori (H. pylori) infection was evaluated by bacterial density of tissue implants and inflammatory responses by histology. The specimens were taken from gastric angle and antrum (greater and lesser curvature sides) by gastroduodenal endoscopy. In urease test, the severity was measured in 3 grades according to color change of the agar: those change are within 30 minutes (grade 3), 30 minutes to 3 hours (grade 2), and 3 to 6 hours (grade 1), respectively; while the grade 0 indicated no color change occurring 6 hours after tissue inoculation. The severity of infection was assessed according to the bacterial density under high power microscopic fields (Gram's stain). Grade 0 indicated no bacterium seen; grade 1, only 1 to 10 bacteria at all fields; grade 2, 1 to 3 bacteria in each high power field; and grade 3 was 4 bacteria or more on average in each high power field. The degree of inflammatory response was evaluated by inflammatory cell infiltration (H & E stain) and classified into grade 0, 1 and 2, which indicated the inflammatory cell infiltration below 50%, between 50% and 75%, and above 75%, respectively. There are no positive relationships among urease test reaction time, bacterial density grading and degrees of inflammatory cell infiltration. Clinically, the reaction time of urease test cannot reflect the severity of H. pylori infection semi-quantitatively, either in terms of bacterial density or cellular inflammatory response.

Adult↗

PubMed1995

Does prediction of epitopes from the primary structure of a protein represent the epitope in the native structure? A study using cobrotoxin.

In contrast to observations made with S. aureus V8 protease-digest hydrolysates, the antigenic structures of reduced and S-carboxymethylated (RCM)-cobrotixin were notably affected following hydrolysis of RCM-cobrotoxin with chymotrypsin. The peptide separated from the V8 protease-digest hydrolysates with a sequence at positions 22-38 of cobrotoxin exhibited a nearly equal reactivity toward the anti-RCM-cobrotoxin antibodies as RCM-cobrotoxin. Chymotryptic cleavage on this segment caused a precipitous drop in the antigenicity of RCM-cobrotoxin. Alternatively, the N-terminal and C-terminal regions of RCM-cobrotoxin encompassed other antigenic determinants which exhibited low reactivities toward anti-RCM-cobrotoxin antibodies. The epitope structures of RCM-cobrotoxin are in line with those predicted from the hydrophobicity profile of cobrotoxin, but the notably immunoreactive region in the C-terminal region of native toxin molecule (Ref. 1) cannot be predicted from analysis of its primary structure. Moreover, RCM-cobrotoxin had a superior reactivity toward anti-RCM-cobrotoxin antibodies than cobrotoxin did. These results indicate that the epitope structures in RCM-cobrotoxin and cobrotoxin are different.

Antibodies↗

PubMed1994

Functional involvement of Lys-6 in the enzymatic activity of phospholipase A2 from Bungarus multicinctus (Taiwan banded krait) snake venom.

Phospholipase A2 (PLA2) from Bungarus multicinctus snake venom was subjected to Lys modification with 4-chloro-3,5-dinitrobenzoate and trinitrobenzene sulfonic acid, and one major carboxydinitrophenylated (CDNP) PLA2 and two trinitrophenylated (TNP) derivatives (TNP-1 and TNP-2) were separated by high-performance liquid chromatography. The results of amino acid analysis and sequence determination revealed that CDNP-PLA2 and TNP-1 contained one modified Lys residue at position 6, and both Lys-6 and Lys-62 were modified in TNP-2. It seemed that the Lys-6 was more accessible to modified reagents than other Lys residues in PLA2. Modification of Lys-6 caused a 94% drop in enzymatic activity as observed with CDNP-PLA2 and TNP-1. Alternatively, the enzyme modified on both Lys-6 and Lys-62 retained little PLA2 activity. Either carboxydinitrophenylation or trinitrophenylation did not significantly affect the secondary structure of the enzyme molecule as revealed by the CD spectra, and Ca2+ binding and antigenicity of Lys-6-modified PLA2 were unaffected. Conversion of nitro groups to amino groups resulted in a partial restoration of enzymatic activity of CDNP-PLA2 to 32% of that of PLA2. It reflected that the positively charged side chain of Lys-6 might play an exclusive role in PLA2 activity. The TNP derivatives could be regenerated with hydrazine hydrochloride. The biological activity of the regenerated PLA2 is almost the same as that of native PLA2. These results suggest that the intact Lys-6 is essential for the enzymatic activity of PLA2, and that incorporation of a bulky CDNP or TNP group on Lys-6 might give rise to the distortion of the interaction between substrate and the enzyme molecule, and the active conformation of PLA2.

Amino Acid Sequence↗

PubMed1994

The essentiality of B chain in stabilizing the structure of the A chain in beta 1-bungarotoxin from Bungarus multicinctus venom.

The dynamic of Trp residue in beta 1-bungarotoxin (beta 1-Bgt), the A chain of beta 1-Bgt and phospholipase A2 (PLA2) was assessed by fluorescence measurement. Acrylamide quenching studies showed that the exposure degree of the Trp in PLA2 is higher than the Trp in beta 1-Bgt. The Trp of beta 1-Bgt had a higher accessibility for iodide, reflecting that the basic nature of the B chain might exert an attractive electrostatic force for iodide and increase the susceptibility of Trp in the A chain to iodide. Removal of the B chain of beta 1-Bgt did not significantly affect the exposure degree of Trp in the A chain. Alternatively, the polarity of the environment around the Trp and the hydrophobic character of ANS and substrate binding sites in the separated A chain changed. Measurement of Trp fluorescence with increasing temperature showed that the stability of structure of beta 1-Bgt was higher than those of the separated A chain and PLA2. These results suggest that the B chain might interact with the A chain and stabilize the conformation of the A chain in beta 1-Bgt.

Animals↗

PubMed1994

Comparison of mutation spectra induced by N-ethyl-N-nitrosourea in the hprt gene of Mer+ and Mer- diploid human fibroblasts.

N-Ethyl-N-nitrosourea (ENU) forms several major adducts upon reaction with DNA, of which ethylation at the O6 position of guanine and the O4, O2 and N3 positions of thymine have been implicated to be mutagenic lesions. To investigate what specific kinds of ENU-induced mutations were affected by the repair ability of O6-alkylguanine-DNA alkyltransferase (AGT), we examined the mutations in the hypoxanthine (guanine) phosphoribosyltransferase gene (hprt) in 87 independent mutants derived from ENU-treated AGT proficient (Mer+) or deficient (Mer-) diploid human fibroblasts. Of the characterized mutations, 97% were single base substitutions. The major difference in the mutation spectra was that the frequency of G.C to A.T transitions was significantly higher in Mer- mutants (16/38) than in Mer+ mutants (4/33). The results indicate that AGT removes O6-ethylguanine, thus protecting human cells from parts of the cytotoxic and mutagenic effects of ENU. A high frequency of T.A to A.T transversions induced by ENU was observed in both Mer+ (52%) and Mer- (34%) mutants. This type of mutation was less frequently observed (10%) in N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced mutants derived from the same Mer+ cells in our previous report (J. Mol. Biol., 221, 421, 1991). Comparison of alkylating lesions formed by MNNG and ENU indicates that O2-ethylthymine and N3-ethylthymine are potent mutational adducts for T to A transversions. The occurrence of ENU-induced T.A base pair transversions showed a strong strand bias; 35/37 were located on the non-transcribed strand, assuming thymine is the mutagenic lesion. The result suggests a difference in repair capacity of ethylthymine on the two strands. In addition, this type of mutation preferentially occurred at 5'-Pu-T sequences.

Base Sequence↗

PubMed1994

Mutations of the p53 gene in human functional adrenal neoplasms.

To clarify gene alterations in functional human adrenal tumors, we performed molecular analysis for p53 abnormalities in 23 cases with adrenal neoplasms. The immunohistochemical study with anti-p53 monoclonal antibody pAb1801 demonstrated that 10 of 23 (43.5%) cases overexpressed p53 protein in the tumor cells. Using a polymerase chain reaction-single strand conformation polymorphism study, 5 of 6 (83.3%) pheochromocytoma tissues (1 malignant and 5 benign) and 11 of 15 (73.3%) adrenocortical adenomas (2 with Cushing's syndrome and 13 with primary aldosteronism, all benign) showed an apparent electrophoretic mobility shift between the tumor and its paired adjacent normal adrenal tissue. Such differences were detected in exon 4 (12 cases), exon 5 (2 cases), and exon 7 (3 cases). The types of these mutations in exon 4 were a substitution from threonine (ACC) to isoleucine (ATC) at codon 102 in 5 cases, from glutamine (CAG) to histidine (CAC) at codon 104 in 1 case, from glycine (GGG) to alanine (CGG) at codon 117 in 1 case, from glutamate (GAG) to glutamine (CAG) at codon 68 in 1 case, and single base changes resulting in a premature stop codon at codon 100 in 2 cases. A 2-basepair deletion at codon 175 in exon 5 resulting in a frame shift was identified in 1 case. A single point mutation was identified, resulting in the substitution of glutamine (CAG) for arginine (CGG) at codon 248 of exon 7 in 1 case. A single basepair deletion at codon 249 resulted in a frame shift in 2 cases. There was 1 case with malignant pheochromocytoma that combined a single point mutation in exon 4 and a single base deletion in exon 7. Only 2 of 23 cases showed a loss of a normal allele encoding in the p53 gene. Northern blot analysis with 1.8-kilobase p53 cDNA revealed that p53 mRNA was overexpressed in 6 cases. Our results indicate that high frequencies of p53 gene mutation, especially in exon 4, exist in functional adrenal tumors. As p53 protein is a regulator of guanine nucleotide synthesis, the loss of normal inhibitory regulation by the p53 mutation would serve to increase the availability of GTP for the transduction of signals essential for increased cell growth and hormone expression in the adrenal tumors. These findings suggest that the p53 gene mutation may play a role in the tumorigenesis of benign and functional human adrenal tumors.

Adenoma↗

PubMed1994

Brain natriuretic peptide is synthesized in the human adrenal medulla and its messenger ribonucleic acid expression along with that of atrial natriuretic peptide are enhanced in patients with primary aldosteronism.

The present study was designed to determine whether brain natriuretic peptide (BNP) is synthesized in the human adrenal gland and, if so, to investigate the BNP content of adrenal tissue and the changes in BNP messenger ribonucleic acid (mRNA) in patients with primary aldosteronism. A considerable amount of BNP-like immunoreactive substances was extracted from the adrenal glands of kidney donors for transplantation (0.21 +/- 0.02 pmol/g wet tissue; n = 3) and the remnant nontumorous adrenal glands of patients with primary aldosteronism (0.20 +/- 0.05 pmol/g wet tissue; n = 3; mean +/- SEM). Immunohistochemical study with a specific antihuman BNP antibody revealed that BNP-like immunoreactivity was localized in the adrenal medullary area, and an in situ hybridization study indicated that the BNP mRNA was mainly expressed in the cells of adrenal medulla. Using a reverse transcription and polymerase chain reaction technique, BNP complementary DNA was cloned from the human adrenal gland, and the sequence was identical to that of BNP identified in the atria. The level of BNP mRNA in the adrenal glands of patients with primary aldosteronism (n = 4) was obviously elevated compared to that in the kidney donors (n = 4), as determined by Northern blot analysis. Quantitative polymerase chain reaction measurements of BNP and atrial natriuretic peptide (ANP) mRNAs showed that both of the adrenomedullary natriuretic peptide gene transcriptions were enhanced in patients with primary aldosteronism, but the amount of ANP mRNA was far higher than that of BNP mRNA in the human adrenal gland. Our results are the first to indicate that BNP is synthesized in the human adrenal medulla, and that such medullary BNP synthesis increases in patients with primary aldosteronism. These facts support the proposal that adrenomedullary BNP along with ANP may play some role in water and electrolyte homeostasis or act in a paracrine manner to regulate adrenocortical functions.

Adrenal Medulla↗

PubMed1994

Long-term follow-up and serological study after triple therapy of Helicobacter pylori-associated duodenal ulcer.

OBJECTIVE: The purpose of this study was to monitor the change of IgG antibodies to Helicobacter pylori during the course of a long-term follow-up after the eradication of H. pylori, as well as to evaluate the efficacy of triple therapy in preventing the recurrence of duodenal ulcer (DU). METHODS: Twenty-three DU patients with positive H. pylori were studied; among those patients, 20 ulcers healed after initial treatment of triple therapy. Each patient received a follow-up endoscopy and biopsy examination every 3 months for 1 yr, or whenever the ulcer symptoms recurred. Serological tests for IgG antibodies were performed with a commercial ELISA. RESULTS: Of the 20 patients with healed ulcers, one dropped out of this study; the recurrence rate of DU at 1 yr was 5% (1/19). There was no recurrence of ulcer in 17 H. pylori-negative patients and only one recurrence out of two H. pylori-positive patients. After the eradication of H. pylori, the seroconversion rates of IgG were 5%, 21%, 42%, 76%, and 94% at 0, 3, 6, 9, and 12 months, respectively. After 6 months, 95% (18/19) of the patients in which H. pylori was eradicated had at least a one-grade fall in IgG titers (p < 0.001). One patient had a relapse of H. pylori which was accompanied with re-elevating IgG titers. CONCLUSIONS: We conclude that seroconversion, which usually occurs after 1 yr, indicates complete eradication of H. pylori. Furthermore, we suggest that the success of the eradication of H. pylori can be judged without endoscopic examination, 8 months after treatment by triple therapy, according to the fall of IgG titers or seroconversion. Re-elevating IgG titers may be a warning of the possibility of relapse or reinfection of H. pylori.

Anti-Ulcer Agents↗

PubMed1993

Simultaneous determination of Zn, Cd, Pb, and Cu in urine of patients with blackfoot disease using anodic stripping voltammetry.

Blackfoot disease (BFD) is an endemic peripheral vascular disorder resulting in gangrene of the lower extremities, especially the feet, among residents in a limited area on the southwest coast of Taiwan. In the present study, the concentrations of zinc, cadmium, lead, and copper in urine of BFD patients with matched normal controls are investigated by differential pulse anodic stripping voltammetry (DPASV) on a hanging mercury drop electrode (HMDE). The analytical results indicate that urinary copper, cadmium, and lead of the BFD patients are significantly higher than those of the controls. In addition, the patients showed a significantly lower concentration of zinc in the urine than the normal controls. The possible connection of these elements with the etiology of the disease is discussed.

Electrochemistry↗

PubMed1993

Increased adrenal medullary atrial natriuretic polypeptide synthesis in patients with primary aldosteronism.

The present study was designed to determine whether atrial natriuretic polypeptide (ANP) is synthesized in the human adrenal gland and, if so, to investigate the ANP content of adrenal tissue and the ANP mRNA changes in patients with primary aldosteronism. A considerable amount of human alpha ANP-like immunoreactive substances was extracted from the remnant adrenal glands of three patients with primary aldosteronism (1.44, 1.0, and 0.77 pmol/g wet tissue; mean +/- SD, 1.07 +/- 0.28 pmol/g) and the adrenal glands of three kidney donors for transplantation (0.93, 0.58, and 0.27 pmol/g wet tissue; mean +/- SD, 0.59 +/- 0.27 pmol/g). High performance gel permeation chromatographic analysis coupled with a RIA of the tissue extract showed that the molecular form of ANP in the adrenal gland was the precursor form, i.e. human gamma ANP. An in situ hybridization study using an ANP cRNA probe indicated that the ANP mRNA was localized mainly in the medullary area of the gland. Northern blot analysis, using ANP cDNA as a probe, detected ANP mRNA in the adrenal gland. Furthermore, the level of ANP mRNA in the adrenal glands of patients with primary aldosteronism was obviously elevated compared to that in the kidney donors. Our results were the first to indicate that ANP is synthesized in the human adrenal medulla, and such medullary ANP synthesis increases in patients with hypermineralocorticoidism. These facts support the proposal that extraatrial (medullary) ANP synthesis might act in a paracrine or endocrine manner to regulate water and electrolyte homeostasis.

Adrenal Glands↗

PubMed1993

Eradication of Helicobacter pylori infection and the recurrence of duodenal ulcers.

The purpose of this study was to compare the performance of different regimens on Helicobacter pylori (H. pylori) eradication and duodenal ulcer recurrence. During a four-week period, 59 patients with duodenal ulcers who were positive for H. pylori infection were randomly treated with one of three regimens. Seventeen patients were treated with ranitidine, 19 with colloidal bismuth subcitrate (CBS), and 23 with triple therapy (CBS, tetracycline and metronidazole). Forty-six patients with healed ulcers after treatment received follow-up for six months without maintenance therapy. The recurrence rates of duodenal ulcers confirmed by endoscopy in these three groups were 64%, 33% and 0% at the third month, and 73%, 67% and 5% at the sixth month, respectively. In the ranitidine therapy group, H. pylori infection was still present at the final follow-up. In the CBS therapy group, H. pylori was suppressed initially, but recurred in all cases. In the triple therapy group, there was only one case in which H. pylori infection persisted and where ulcer recurrence occurred after 3.5 months. The remaining cases were all H. pylori negative and had no recurrence of duodenal ulcers during the six months of follow-up. Overall, 19/27 (70%) patients positive for H. pylori had a recurrence of duodenal ulcers, while none of the 19 patients who were negative for H. pylori had a recurrence of ulcers at the sixth month. This study shows that triple therapy is more effective than the other two regimens in the eradication of H. pylori and in reducing the recurrence of ulcers. H. pylori may play a role in the recurrence of the duodenal ulcer.

Adolescent↗

PubMed1993

Characterization of R plasmids from Shigella sonnei in Taiwan.

Plasmids of 70MDa from 6 Shigella sonnei isolates originating in Kaohsiung, Taiwan were found. All of them encoded resistance to chloramphenicol and tetracycline. These plasmids belonged to the H II incompatibility group. The fertility inhibition property of plasmids was the fi- type. Restriction endonuclease analysis revealed the six plasmids all had identical restriction patterns with BamH I, Hind III and Nru I. All six plasmids carried the type I chloramphenicol acetyltransferase gene and the class B tetracycline resistance gene as determined by Southern hybridization with DNA probes of various antimicrobial resistance genes. The results demonstrated that S. sonnei isolates harboured the same R plasmid.

Chloramphenicol O-Acetyltransferase↗

PubMed1993

[Study on urinary arsenic, lead and cadmium concentrations in a steel production workers].

Lead and cadmium levels in urine specimens were determined simultaneously by polarographic method, and arsenic by hydride atomic absorption spectrophotometry. The urine specimens were pretreated with a mixed acid solution and ORTHO bilevel urine metal control and APG Setpoint standard were used to check the analytical accuracy. The results showed the total arsenic [As], lead [Pb], cadmium [Cd] levels in the production and the quality control workers, were significantly higher than those in the control. The results indicate the need for an immediate improvement in workplace ventilation and industrial hygiene practices.

Arsenic↗

PubMed1993

Chemical modification of amino groups in cardiotoxin III from Taiwan cobra Naja naja atra) venom.

Cardiotoxin III (CTX III), a major cardiotoxin analogue isolated from the Taiwan cobra (Naja naja atra) venom was modified, either with trinitrobenzene sulfonate (TNBS) or 4-chloro-3,5-dinitrobenzoate (CDNB). Under the conditions of limited reagent availability, three mono-TNP derivatives modified at Lys-5, 12, or 44, and three mono-CDNP derivatives at Lys-12, 23, or 44 were isolated, respectively. The biological activities of CTX III were more or less affected after each of these reactive amino groups were modified. In particular, the hemolytic activity to human erythrocytes and cytotoxicity on NS-1 cells of CTX III decreased to 31% and 50%, respectively, when Lys-12 was trinitrophenylated. More pronounced alteration in these activities was observed as this amino group was carboxydinitrophenylated. A good correlation between the hemolytic activity and cytotoxicity was found. These results indicate that epsilon-amino group at Lys-12 is most closely related to the hemolytic and cytotoxic activities of CTX III. The antigenicity of modified derivatives still remained intact as measured by ELISA.

Animals↗

PubMed1993

Characterization of genetic defects of hemophilia A in patients of Chinese origin.

The molecular characterization of hemophilia A of Chinese origin was carried out by the polymerase chain reaction (PCR) and direct sequencing of patients' factor VIII genes. Single-strand conformation polymorphism (SSCP) and dideoxy fingerprinting (ddF) were used as screening methods to detect mutated DNAs. A total of 102 individuals from 87 different families, including 10 patients (10 families) with mild-to-moderate and 92 patients (77 families) with severe hemophilia A, were analyzed by PCR-SSCP and PCR-ddF. Of the 87 independent cases, 40 revealed a single mutation in the coding regions of their factor VIII genes. These mutations include 21 with single base changes resulting in 8 nonsense and 13 missense codons, 16 with deletion or insertion of 1-11 nucleotides, and 3 with deletion of large DNA fragments. The frequency of 8 of the identified factor VIII polymorphisms or silent mutations was also determined among Chinese. The frequencies for codons 1241, 1269, and 2223 (the numbering system follows J. Gitschier et al., 1984, Nature 312: 326-330) were found to be different from those reported for other populations. As for the 47 severe cases whose mutational events were not readily detected by PCR-SSCP and PCR-ddF, the reverse transcriptase PCR method was applied. In 24 such cases analyzed, 17 were found to be of the "intron 22 mutations" as described by Naylor et al. (1992, The Lancet, 342: 1066-1067), accounting for 39% of Chinese patients with hemophilia A.

Amino Acid Sequence↗