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Biomedical subjects

H J Chang

Publications and source records attributed to H J Chang.

At least 19 recordsLinked to original sources
PubMed2005

Pre-operative chemo-radiotherapy improves the sphincter preservation rate in patients with rectal cancer located within 3 cm of the anal verge.

AIMS: To evaluate whether pre-operative chemo-radiotherapy (CRT) improves the sphincter preservation rate for distal rectal cancers within 3 cm of the anal verge. METHODS: Between January 2001 and December 2004, 49 patients underwent surgery with or without pre-operative CRT for primary rectal adenocarcinoma within 3 cm of the anal verge. Clinical data were retrospectively reviewed, including stage workups, surgical records and pathology records to determine sphincter preservation rate and the factors influencing sphincter preservation. RESULTS: Of 49 patients with rectal tumours within 3 cm of the anal verge, 31 underwent pre-operative CRT followed by surgery (CRT group), and 18 underwent surgery alone (non-CRT group). Sphincter preservation was possible in 11 of 31 CRT patients, and only one of 18 non-CRT patients (p=0.036). The factors most influencing sphincter preservation were reduction in tumour size (p=0.005) and downstaging (p=0.001) following pre-operative CRT. CONCLUSION: We could observe that sphincter preservation was improved in CRT group with statistical significance when compared to non-CRT group in our study patients with rectal cancer within 3 cm of the anal verge.

Adenocarcinoma↗
PubMed2005

Comparative study of the subserosal versus submucosal dye injection method for sentinel node biopsy in gastric cancer.

AIMS: To determine the most efficient injection method for a sentinel node (SN) biopsy in gastric cancer. METHODS: Gastric adenocarcinoma patients without serosal invasion and distant metastasis were prospectively enrolled in this study. Isosulfan blue was injected into the subserosa (SS) of the first set of 71 consecutive patients and an intraoperative endoscopic submucosal (SM) injection of the same dye was injected into the second set of 50 consecutive patients. After the biopsy of blue-stained SNs, a gastrectomy with a D2 lymphadenectomy was performed. All dissected lymph nodes were evaluated for metastasis. The results of the SN biopsies were compared between the SS and SM dye injection methods. RESULTS: Detection rate (0.92 vs 0.94), mean number of SNs (2.5 vs 2.9) and sensitivity (0.61 vs 0.46) of the SN biopsies were not significantly different between the SS and SM injection methods (P>0.05). The operation time was significantly shorter in the SS than the SM injection method (159.7 vs 172.7 min, P=0.030). CONCLUSIONS: Both injection methods were equally efficient in their roles for a SN biopsy in gastric cancer. However, the SS injection method was more preferable due to its easy technique and short operation time.

Adenocarcinoma↗
PubMed2005

Tuberculin reactivity in adults after 50 years of universal bacille Calmette-Guerin vaccination in Taiwan.

We aimed to assess whether tuberculin reactivity in adults is affected by bacille Calmette-Guerin (BCG) vaccination after 50 years of universal BCG vaccination with 80-95% coverage. A community-based study on tuberculin reactivity in 619 participants was conducted in February 2000 in Keelung city, Taiwan. Information on BCG vaccination policies and annual risk of infection (ARI) in the underlying population was extracted from consecutive national prevalence surveys relating to the period 1952-1997. Compared with the expected ARI estimate, the standardized morbidity ratio of positive tuberculin response for vaccination in infancy was 2.2 (95% CI 0.3-15.5) for those aged <10 years. The corresponding figures for older age groups ranged from 3.6 (95% CI 2.2-5.9) for those aged 10-12 years to 0.7 (95% CI 0.5-0.9) for those aged 57-67 years. This suggests that the effect of BCG vaccination on positive tuberculin response in adults aged >30 years is probably negligible irrespective of age at vaccination or revaccination and that the tuberculin skin test can be used to diagnose TB in control programmes in countries with moderate or high incidence of TB.

Adolescent↗
PubMed2004

Enhanced optical nonlinearity near the photonic bandgap edges of a cholesteric liquid crystal.

The third-order Kerr nonlinear optical effect of a one-dimensional photonic bandgap structure of a cholesteric liquid crystal is investigated. In a femtosecond nonlinear transmission measurement, nonlinear optical changes in the bandgap edges are observed. From analysis of the dispersion relation, Kerr nonlinear coefficients of nematics, forming the cholesteric liquid crystal, are found to be enhanced by 1-2 orders of magnitude through the photonic bandgap structure.

Journal Article↗
PubMed2001

Crystallization and preliminary crystallographic results of apo and complex forms of human dehydroepiandrosterone sulfotransferase.

Dehydroepiandrosterone sulfotransferase converts dehydroepiandrosterone (DHEA) and some other steroids to their sulfonated forms. The human enzyme has been crystallized in the presence of substrate (DHEA) alone, in the presence of substrate and non-sulfated cofactor analogue (PAP) and in the absence of both substrate and PAP in our laboratory, with data sets collected at a synchrotron source. The crystals of the uncomplexed form belong to the orthorhombic space group C222(1), with unit-cell parameters a = 85.26, b = 87.69, c = 108.20 A and data 99.2% complete to 2.35 A resolution. The DHEA complex crystallizes in the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 74.46, b = 127.49, c = 44.59 A and data 92.9% complete to 2.15 A resolution. The ternary complex crystallizes in the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 62.25, b = 87.28, c = 138.86 A and data 98.6% complete to 2.50 A resolution. Preliminary molecular-replacement solutions indicate significant variations in dimer formation.

Apoenzymes↗
PubMed2001

Mechanisms of diethylstilbestrol-induced calcium movement in MG63 human osteosarcoma cells.

The effect of the estrogen diethylstilbestrol (DES) on cytosolic free Ca(2+) levels ([Ca(2+)](i)) in MG63 human osteoblasts was explored by using fura-2 as a Ca(2+) indicator. DES at concentrations between 5--20 microM induced an immediate increase in [Ca(2+)](i) in a concentration-dependent manner with an EC(50) of 10 microM. Removing extracellular Ca(2+) reduced the Ca(2+) signal by 70%. Pretreatment with 50 microM La(3+) or 10 microM of nifedipine, verapamil and diltiazem did not change 20 microM DES-induced [Ca(2+)](i) increases. Addition of 3 mM Ca(2+) increased [Ca(2+)](i) in cells pretreated with 20 microM DES in Ca(2+)-free medium. Pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor) to deplete the endoplasmic reticulum Ca(2+) store partly inhibited 20 microM DES-induced Ca(2+) release, but addition of carbonylcyanide m-chlorophenylhydrazone (CCCP; a mitochondrial uncoupler) and thapsigargin together abolished DES-induced Ca(2+) release. Conversely, pretreatment with 20 microM DES abrogated CCCP- and thapsigargin-induced Ca(2+) release. Inhibition of phospholipase C activity with 2 microM U73122 did not alter 20 microM DES-induced Ca2+ release. Another estrogen 17beta-estradiol also increased [Ca(2+)](i) in a concentration-dependent manner with an EC50 of 7 microM. Together, the data indicate that in human osteoblasts, DES increased [Ca(2+)](i) via causing Ca(2+) release from both mitochondria and the endoplasmic reticulum in a phospholipase C-independent manner, and by causing Ca(2+) influx.

Calcium↗
PubMed2001

Solution structure, backbone dynamics, and stability of a double mutant single-chain monellin. structural origin of sweetness.

Single-chain monellin (SCM), which is an engineered 94-residue polypeptide, has been characterized as being as sweet as native two-chain monellin. Data from gel-filtration high performance liquid chromatography and NMR has proven that SCM exists as a monomer in aqueous solution. In order to determine the structural origin of the taste of sweetness, we engineered several mutant SCM proteins by mutating Glu(2), Asp(7), and Arg(39) residues, which are responsible for sweetness. In this study, we present the solution structure, backbone dynamics, and stability of mutant SCM proteins using circular dichroism, fluorescence, and NMR spectroscopy. Based on the NMR data, a stable alpha-helix and five-stranded antiparallel beta-sheet were identified for double mutant SCM. Strands beta1 and beta2 are connected by a small bulge, and the disruption of the first beta-strand were observed with SCM(DR) comprising residues of Ile(38)-Cys(41). The dynamical and folding characteristics from circular dichroism, fluorescence, and backbone dynamics studies revealed that both wild type and mutant proteins showed distinct dynamical as well as stability differences, suggesting the important role of mutated residues in the sweet taste of SCM. Our results will provide an insight into the structural origin of sweet taste as well as the mutational effect in the stability of the engineered sweet protein SCM.

Arginine↗
PubMed2001

Acute and long-term outcomes of stenting in coronary vessel > 3.0 mm, 3.0-2.5 mm, and < 2.5 mm.

We compared the acute and long-term outcomes of stentings in coronary vessels > 3.0 mm, 3.0-2.5 mm, and < 2.5 mm. A total of 1,152 patients underwent coronary stenting was divided into three groups based on the reference vessel size. Group A consisted of 598 patients (667 lesions) with a reference vessel diameter > 3.0 mm, group B 485 patients (544 lesions) with a reference vessel diameter of 3.0-2.5 mm, and group C 114 patients (119 lesions) with a reference vessel diameter < 2.5 mm. The procedural success, stent thrombosis, and in-hospital cardiac event rate were similar in the three groups. At 6-month angiographic follow-up, the lesion restenotic rate was significantly higher in the small-vessel group (14%, 22%, and 26% in groups A, B, and C, respectively; P = 0.011). These differences appeared to result from a lesser acute gain and a lesser net gain in small-vessel group; the late luminal loss was similar in the three groups. During a follow-up duration of 28 +/- 3 months, group C patients had a significantly lower rate of event-free survival than the group A and B patients (71% vs. 85% and 82%; P = 0.002). Stepwise regression analysis demonstrated that complex lesion (P = 0.032) and long lesion (P = 0.046) are independent predictors of restenosis in very-small-vessel (< 2.5 mm) stenting. In conclusion, the acute results of stenting in small coronary arteries appear safe and feasible with a high procedural success rate and a low incidence of stent thrombosis. Stenting in patients with a small coronary artery appears to have a similar in-hospital cardiac event rate, but a higher angiographic restenosis rate and a lower event-free survival rate, compared to stenting in patients with a larger coronary artery. The predictors of restenosis in very-small-vessel stenting are complex lesions and long lesions. Cathet Cardiovasc Intervent 2001;53:314-322.

Aged↗
PubMed2001

Human dehydroepiandrosterone sulfotransferase: purification and characterization of a recombinant protein.

Dehydroepiandrosterone sulfate is the most abundant sulfated steroid transformed in human tissues and serves as a precursor for steroid hormones. Recombinant human dehydroepiandrosterone sulfotransferase (DHEA-ST) expressed in glutathione sulfotransferase fusion form in E. coli was purified using glutathione sepharose 4B affinity adsorption chromatography, a Factor Xa cleavage step, and Q-sepharose fast flow column chromatography. The homogeneous preparation had an activity toward dehydroepiandrosterone (DHEA) of 150+/-40 nmol/min per mg of protein under the assay conditions at an overall yield of 38.4%. The recombinant human DHEA-ST was shown to have a subunit mass of 34 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, while having a molecular mass of 67.2 kDa by Superose-12 gel filtration. Our results indicate that the active recombinant enzyme expressed in E. coli is a homodimer.Biochemical properties for purified DHEA-ST were studied using DHEA as a substrate. The optimum pH ranged from pH 7 to 8, and the optimum temperature 40-45 degrees C. Ninety percent of basal DHEA-ST activity remained even after the enzyme was treated at 45 degrees C for 15 min. The 50% inactivation concentration of NaCl for DHEA-ST activity was determined to be around 500 mM. The K(m) value for DHEA was 1.9+/-0.3 microM and V(max)=190+/-18 nmol/min per mg of protein at 37 degrees C, pH 7.5.

Chromatography, Affinity↗
PubMed2001

Nordihydroguaiaretic acid elevates osteoblastic intracellular Ca2+.

Nordihydroguaiaretic acid (NDGA) is widely used as a pharmacological tool to inhibit lipoxygenases; however, recent evidence suggests that it increases renal intracellular [Ca2+]i via novel mechanisms. Here the effect of NDGA on Ca2+ signaling in MG63 osteoblastic cells was explored using fura-2 as a Ca2+ indicator. NDGA (2-50 microM) increased [Ca2+]i in a concentration-dependent manner. The signal comprised an initial rise and an elevated phase over a time period of 4 min. Removing extracellular Ca2+ reduced 2-50 microM NDGA-induced signals by 62+/-2%. After incubation with 50 microM NDGA in Ca2+-free medium for several minutes, addition of 3 mM CaCl2 induced an increase in [Ca2+]i. NDGA (50 microM)-induced [Ca2+]i increases were not changed by pretreatment with 10 microM of verapamil, diltiazem, nifedipine, nimodipine and nicardipine. In Ca2+-free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin (1 microM) inhibited 50 microM NDGA-induced [Ca2+]i increases by 69+/-3%. Inhibition of phospholipase C with 2 microM U73122 had little effect on 50 microM NDGA-induced Ca2+ release. Several other lipoxygenase inhibitors had no effect on basal [Ca2+]i. At a concentration that did not increase basal [Ca2+]i, NDGA (1 microM) did not alter 10 microM ATP- or 1 microM thapsigargin-induced [Ca2+]i increases. Alteration of protein kinase C activity with 1 nM phorbol 12-myristate 13-acetate or 2 microM GF 109203X did not affect 50 microM NDGA-induced [Ca2+]i increases. Together, the results show that NDGA increased [Ca2+]i in osteoblasts in a lipoxygenase-independent manner, by releasing stored Ca2+ in a fashion independent of phospholipase C activity, and by causing Ca2+ influx.

Calcium↗
PubMed2001

Mechanism of bradykinin-induced Ca(2+) mobilization in MG63 human osteosarcoma cells.

BACKGROUND: The effect of bradykinin on intracellular free Ca(2+) levels ([Ca(2+)](i)) in MG63 human osteosarcoma cells was explored using fura-2 as a Ca(2+) dye. METHODS/RESULTS: Bradykinin (0.1 nM-1 microM) increased [Ca(2+)](i) in a concentration-dependent manner with an EC(50) value of 0.5 nM. The [Ca(2+)](i) signal comprised an initial peak and a fast decay which returned to baseline in 2 min. Extracellular Ca(2+) removal inhibited the peak [Ca(2+)](i )signals by 35 +/- 3%. Bradykinin (1 nM) failed to increase [Ca(2+)](i) in the absence of extracellular Ca(2+ )after cells were pretreated with thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor; 1 microM). Bradykinin (1 nM)-induced intracellular Ca(2+) release was nearly abolished by inhibiting phospholipase C with 2 microM 1-(6-((17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). The [Ca(2+)](i )increase induced by 1 nM bradykinin in Ca(2+)- free medium was abolished by 1 nM HOE 140 (a B2 bradykinin receptor antagonist) but was not altered by 100 nM Des-Arg-HOE 140 (a B1 bradykinin receptor antagonist). Pretreatment with 1 pM pertussis toxin for 5 h in Ca(2+) medium inhibited 30 +/- 3% of 1 nM bradykinin-induced peak [Ca(2+)](i) increase. CONCLUSIONS: Together, this study shows that bradykinin induced [Ca(2+)](i) increases in a concentration-dependent manner, by stimulating B2 bradykinin receptors leading to mobilization of Ca(2+) from the thapsigargin-sensitive stores in a manner dependent on inositol-1,4,5-trisphosphate, and also by inducing extracellular Ca(2+) influx. The bradykinin response was partly coupled to a pertussis toxin-sensitive G protein pathway.

Biological Transport↗
PubMed2001

Critical residues for the specificity of cofactors and substrates in human estrogenic 17beta-hydroxysteroid dehydrogenase 1: variants designed from the three-dimensional structure of the enzyme.

Human estrogenic 17beta-hydroxysteroid dehydrogenase is an NADP(H)-preferring enzyme. It possesses 11- and 4-fold higher specificity toward NADP(H) over NAD(H) for oxidation and reduction, respectively, as demonstrated by kinetic studies. To elucidate the roles of the amino acids involved in cofactor specificity, we generated variants by site-directed mutagenesis. The results showed that introducing a positively charged residue, lysine, at the Ser12 position increased the enzyme's preference for NADP(H) more than 20-fold. Substitution of the negatively charged residue, aspartic acid, into the Leu36 position switched the enzyme's cofactor preference from NADPH to NAD with a 220-fold change in the ratio of the specificity toward the two cofactors in the case of oxidation. This variant dramatically abolished the enzyme's reductase function and stimulated its dehydrogenase activity, as shown by enzyme activity in intact cells. The substrate-binding pocket was also studied with four variants: Ser142Gly, Ser142Cys, His221Ala, and Glu282Ala. The Ser142Gly variant abolished most of the enzyme's oxidation and reduction activities. The residual reductase activity in vitro is less than 2% that of the wild-type enzyme. However, the Ser142Cys variant was fully inactive, both as a partially purified protein and in intact cells. This suggests that the bulky sulfhydryl group of cysteine entirely disrupted the catalytic triad and that the Ser142 side chain is important for maintaining the integrity of this triad. His221 variation weakened the apparent affinity for estrone, as demonstrated by a 30-fold increase in Michaelis-Menten constant, supporting its important role in substrate binding. This residue may play an important role in substrate inhibition via the formation of a dead-end complex. The formerly suggested importance of Glu282 could not be confirmed.

17-Hydroxysteroid Dehydrogenases↗
PubMed2000

Evaluation of a population-based screening for type 2 diabetes: a community-based screening project in Puli, Taiwan.

BACKGROUND: A Markov method incorporating the relationships between prevalence, incidence, and mortality with respect to type 2 diabetes was used to assess a population-based screening for this disease. METHODS: Data from a population-based screening project for residents of Puli, Taiwan, over 30 years of age (n= 1,219) were used to estimate the annual incidence of asymptomatic type 2 diabetes, the prevalence to incidence (P/I) ratio, and the hazard rate of death due to type 2 diabetes. These parameters were employed to develop a Markov process to evaluate the effects of early detection of type 2 diabetes on the risk of death from this disease in a simulated population (n= 10,000) receiving biennial, 5-year interval, or no screening. RESULTS: The estimated annual incidence, average duration from asymptomatic to symptomatic type 2 diabetes (P/I ratio), and hazard rate for death from this disease were 0.86% (95% CI 0.50-1.48), 10 years (95% CI 7.69-14.01), and 1.1% per year, respectively. This yields an optimal screening interval of 5 years. Simulation of a 5-year interval screening regimen versus no screen ing yielded a relative risk reduction of 31% (95% CI 12-46%). A similar value was found for a biennial screening regime. CONCLUSIONS: The results suggest that early detection of type 2 diabetes via a community-based screening project in developing countries with high prevalence is worthwhile.

Adult↗
PubMed2000

Clinical significance of hepatitis C virus infection to alcoholics with cirrhosis in Korea.

BACKGROUND/METHODS: To investigate the prevalence and clinical significance of hepatitis C virus (HCV) infection and its relationship with the development of hepatocellular carcinoma (HCC), 162 consecutive alcoholic patients with cirrhosis were studied. Alcohol intake and parenteral risk factors were investigated by interview using a questionnaire. All patients had consumed at least 80 g alcohol/day for at least the past 5 years. Sera were tested for anti-HCV using a third-generation enzyme immunoassay (EIA), hepatitis B s antigen (HBsAg), anti-HBs, anti-HBc and anti-HIV. Serum HCV-RNA was detected by a one-tube reverse transcription-polymerase chain reaction (RT-PCR) method. Patients were classified into three groups accroding to the presence or absence of viral markers: (i) cases without anti-HCV or HBsAg (group A); (ii) cases with HBsAg only (group B); and (iii) cases with anti-HCV only (group C). Demographic and clinical findings were compared among the three groups. RESULTS: Anti-HCV was present in 17 cases (10.5%) and HBsAg was present in 47 cases (29%). No patient had both anti-HCV and HBsAg. Group C subjects were the oldest, but the duration of drinking in this group was similar to that of group A. There was no significant difference in the daily alcohol intake among the three groups. Previous surgical operations and tattooing were more frequent in group C. Only one patient in group C was an intravenous drug user. The combined rate of HCC was significantly higher in groups B and C than in groups A (34, 23.5 and 6.1%, respectively). Laboratory data showed a higher platelet count, higher albumin level, lower bilirubin level and lower aspartate aminotransferase/alanine aminotransferase ratio in group C patients than in the other two groups. Hepatitis C virus RNA was detected in 14 of 85 cases tested (16.5%), in 11 of 12 cases (91.7%) with anti-HCV and in three of 73 cases (4.1%) without anti-HCV. CONCLUSIONS: Hepatitis C virus infection is frequent in alcoholic patients with cirrhosis in Korea. Hepatitis C virus, as well as hepatitis B virus, infection may have a synergistic effect on the development of HCC in alcoholic patients.

Adult↗
PubMed2000

Benefits of late coronary artery stenting in patients with acute myocardial infarction with and without thrombolytic therapy.

BACKGROUND: There are no added benefits when balloon angioplasty is conducted in conjunction with thrombolytic therapy in patients with acute myocardial infarction. The purpose of this study was to determine whether or not thrombolysis has an impact on the outcome of late coronary artery stenting following acute myocardial infarction. METHODS: The outcome of late coronary artery stenting in the infarct-related artery following acute infarction was compared in patients with (68 patients, group A) and without (118 patients, group B) prior thrombolytic therapy. RESULTS: The baseline characteristics were similar in the 2 groups except that total occlusion of the infarct-related artery was more common in group B. The angiographic characteristics of the target lesion were similar in the 2 groups; the procedural success rate was 98% in both groups. There was no subacute thrombosis or other complications in either group. The 6-month follow-up coronary angiography and the restenosis rate was 18% in both groups; the reocclusion rate was 2% in group A and 4% in group B. The increment of the left ventricular ejection fraction was similar in both groups (6% versus 7%). During a follow-up duration of 18 +/- 3 months, the mortality rate was 3% versus 2%, reinfarction 0% versus 1%, recurrent angina 6% versus 4%, and target lesion revascularization by angioplasty 13% versus 13% in group A and B patients, respectively. CONCLUSION: The outcome of late coronary artery stenting following acute myocardial infarction in patients with and without prior thrombolytic therapy was comparable. Significant improvement of left ventricular function was noted in both groups.

Adult↗
PubMed1999

Clinical and angiographic outcomes are similar with half, single, or multiple contiguous Palmaz-Schatz stent implantations for a single coronary stenosis.

We compared the immediate and 6-month clinical and angiographic outcomes in patients undergoing a half, a single, or multiple contiguous stent implantations for a single coronary stenosis. Four hundred forty-three consecutive patients, who underwent elective Palmaz-Schatz stent implantations for 542 stenoses between November 1995 and July 1998, were analyzed. Sixty-three patients with 78 stenoses received a half stent (group A), 346 patients with 395 stenoses received a single stent (group B), and 68 patients with 69 stenoses received multiple overlapping stents (group C) for a single coronary stenosis. Seventy-eight half stents were implanted in 78 stenoses in group A, 395 stents in 395 stenoses in group B, and 141 stents in 69 stenoses in group C. The baseline characteristics were similar in the 3 groups. There were no deaths, no subacute thrombosis, and no vascular complications. Forty-nine patients with 57 stenoses in group A, 280 patients with 326 stenoses in group B, and 59 patients with 60 stenosis in group C underwent 6-month follow-up coronary angiography; the restenotic rate per patient was 10% in group A, 20% in group B, and 24% in group C (NS); the restenotic rate per stenosis was 9% in group A, 18% in group B, and 23% in group C (NS). Follow-up of 18 +/- 3 months revealed no differences in mortality, reinfarction, recurrent angina, target narrowing angioplasty, and elective coronary artery bypass surgery among the 3 groups. The overall cardiac event-free survival was 90%, 82%, and 83% in groups A, B, and C, respectively (p = 0.275). Thus, the procedural success rate, the in-hospital morbidity, and the long-term outcome are similar with coronary stenting using a half, a single, or multiple overlapping Palmaz-Schatz stents for a single stenosis.

Angioplasty, Balloon, Coronary↗
PubMed1999

Solution structure of a sweet protein single-chain monellin determined by nuclear magnetic resonance and dynamical simulated annealing calculations.

Single-chain monellin (SCM), which is an engineered 94-residue polypeptide, has proven to be as sweet as native two-chain monellin. SCM is more stable than the native monellin for both heat and acidic environments. Data from gel filtration HPLC and NMR indicate that the SCM exists as a monomer in aqueous solution. The solution structure of SCM has been determined by nuclear magnetic resonance (NMR) spectroscopy and dynamical simulated annealing calculations. A stable alpha-helix spanning residues Phe11-Ile26 and an antiparallel beta-sheet formed by residues 2-5, 36-38, 41-47, 54-64, 69-75, and 83-88 have been identified. The sheet was well defined by backbone-backbone NOEs, and the corresponding beta-strands were further confirmed by hydrogen bond networks based on amide hydrogen exchange data. Strands beta2 and beta3 are connected by a small bulge comprising residues Ile38-Cys41. A total of 993 distance and 56 dihedral angle restraints were used for simulated annealing calculations. The final simulated annealing structures ( k) converged well with a root-mean-square deviation (rmsd) between backbone atoms of 0.49 A for secondary structural regions and 0.70 A for backbone atoms excluding two loop regions. The average restraint energy-minimized (REM) structure exhibited root-mean-square deviations of 1.19 A for backbone atoms and 0.85 A for backbone atoms excluding two loop regions with respect to 20 k structures. The solution structure of SCM revealed that the long alpha-helix was folded into the concave side of a six-stranded antiparallel beta-sheet. The side chains of Tyr63 and Asp66 which are common to all sweet peptides showed an opposite orientation relative to H1 helix, and they were all solvent-exposed. Residues at the proposed dimeric interface in the X-ray structure were observed to be mostly solvent-exposed and demonstrated high degrees of flexibility.

Algorithms↗