Effects of Citrus depressa Hayata juice on high-fat diet-induced obesity in HBV transgenic mice.

The present study investigated the potential anti-obesity properties of Citrus depressa Hayata (CDH) juice in HBV transgenic mice, as well as the impact of fermentation on the effectiveness of the juice. The results revealed that fermentation increased the levels of polyphenols and hesperidin in CDH juice. The animal study demonstrated that both juices were effective in mitigating the weight gain induced by a high-fat diet by correcting metabolic parameter imbalances, reducing hepatic lipid accumulation, and reversing hepatic immune suppression. Furthermore, fermented juice exhibited superior efficacy in managing body weight and inhibiting the expansion of white adipose tissue (WAT). Fermented juice significantly enhanced adiponectin production and PPARγ expression in WAT, while also reducing hypertrophy. This study offers valuable insights into the potential role of CDH juices in combating obesity associated with high fat consumption and underscores the promise of CDH juice as a functional beverage.

The Efficacy of Lactobacillus delbrueckii ssp. bulgaricus Supplementation in Managing Body Weight and Blood Lipids of People with Overweight: A Randomized Pilot Trial.

This study aimed to evaluate the efficacy of Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) in improving body weight, obesity-related outcomes, and lipid profiles of overweight people. Thirty-six overweight participants were randomly assigned to either a probiotic or a placebo group. A placebo powder or L. bulgaricus powder (containing 1 × 108 colony-forming unit (CFU) of the probiotic) was administered daily for 12 weeks. Body composition was determined, and blood tests were performed before and after the intervention. L. bulgaricus supplementation under the present condition did not affect the body weight, fat percentage, or body mass index (BMI) of the participants, while it resulted in a notable decrease in blood triglyceride (TG) levels, which corresponded to a lowering of the TG proportion in the composition of large VLDL (L-XXL sized fractions) and HDL (M and L fractions) in the probiotic-treated group. These results suggest that L. bulgaricus supplementation under the current conditions may not be helpful for losing weight, but it has the potential to decrease blood TG levels by modulating TG accumulation in or transport by VLDL/HDL in obese patients. L. bulgaricus supplements may have health-promoting properties in preventing TG-related diseases in overweight people.

Novel multidisciplinary cooperation model for obstetric medical quality control: A quasi-experimental study.

OBJECTIVE: To explore the impact of a novel multidisciplinary cooperation model in obstetric medical quality control. METHODS: This quasi-experimental study analyzed the quality indicators of full-term pregnant women who underwent vaginal trial labor in Zibo Maternal and Child Health Hospital between July 2021 and June 2022. The pregnant women were divided into two groups based on implementation of novel multidisciplinary cooperation: multidisciplinary and non-multidisciplinary. We compared the rate of labor analgesia, postpartum hemorrhage in vaginal delivery, transfer to cesarean section, and the 5-min Apgar score ≤7 in full-term neonates. RESULTS: A total of 3751 pregnant women were enrolled into the study, of whom 2004 were included in the non-multidisciplinary group and 1747 in the multidisciplinary group. The analgesic rate of delivery of the multidisciplinary group was higher than that of the non-multidisciplinary group (P = 0.000). We established that the rate of postpartum bleeding (P = 0.040), transfer cesarean section (P = 0.003) and the incidence of Apgar score ≤7 in 5 min of full-term neonates (P = 0.038) of the multidisciplinary group was lower than that of the non-multidisciplinary group. There was no significant difference in the mean ages (29.40 ± 3.99 vs. 29.90 ± 4.27 years; P = 0.126), mean delivery gestational ages (39.65 ± 0.87 vs. 39.64 ± 1.06; P = 0.221), mean gravidity values (1.93 ± 1.09 vs. 2.00 ± 1.18; P = 0.586) and mean parity (1.40 ± 0.56 vs. 1.42 ± 0.59; P = 0.635) of the women in the two groups. CONCLUSION: Multidisciplinary cooperation in delivery management can significantly improve some quality indicators. We established the analgesic rate of delivery can be increased and the rate of postpartum bleeding, transfer cesarean section and the incidence of Apgar score ≤7 in 5 min of full-term neonates can be decreased with the implementation of novel multidisciplinary cooperation.

Exposure to incense burning, biomarkers, and the physical health of temple workers in Taiwan.

Incense burning releases heavy particulate matter (PM) and nitrogen dioxide (NO2), known to have adverse effects on human health. Long-term exposure to PM and NO2 increases inflammatory cytokine levels and can induce respiratory diseases. This study examined the association between incense burning exposure and the health status, especially inflammatory biomarkers, of temple workers and volunteers in Taiwan. The longitudinal observational study compared adult temple workers and volunteers, with long-term incense burning exposure, to residents from outpatient clinics in the Chiayi area. Forced expiratory volume in 1 s (FEV1) and serum and exhaled breath condensate (EBC) cytokines were assessed. Nonparametric Mann-Whitney U tests were used to compare cytokine levels of the exposure and control groups during the cold and hot weather seasons. FEV1 was significantly more diminished in the exposed group than in the control group during the cold season. Exposure status was associated with greater hot-cold seasonal differences in serum interleukins (IL)-1ß (regression coefficient (B) = 6.6, 95% confidence interval (CI) = 5.0 to 8.3, p < .001), IL17-A (B = 2.4, 95% CI = 0.3 to 4.5, p = .03), and plasminogen activator inhibitor [PAI]-1 (B = 5.4, 95% CI = 1.5 to 9.3, p = .009). After adjusting for confounders, the groups' serum levels of IL-1ß, IL-17A, and PAI-1 significantly differed. EBC cytokines did not show significant differences. Elevated levels of IL-1ß, IL17-A, and PAI-1 have been associated with various autoinflammatory syndromes and diseases. Given the cultural significance of incense burning, culturally sensitive interventions, including education, policy development, and program implementation, are crucial to protect individuals' health, especially temple workers, from the adverse effects of exposure, addressing the manufacture, importation, and sale of incense.

Impact of prolonged use of adjuvant tocolytics after cervical cerclage on late abortion and premature delivery.

We evaluated the impact of cervical cerclage combined with one or more uterine contraction inhibitors in persistent inhibition of uterine contraction for the treatment of late abortion and premature delivery. This retrospective case series study analysed the medical data of 58 patients who underwent cervical cerclage for cervical insufficiency and simultaneously received one or more uterine contraction inhibitors (indomethacin, ritodrine, and atosiban) and magnesium sulphate at the Zibo Maternal and Child Health Hospital between January 2019 and December 2020.Patients are normal pregnancy who received cervical cerclage without complications. The rate of successful treatment was 74.14% (43/58). The prolonged gestation duration was 16.42 ± 7.84 weeks, and the average delivery gestational age was 35.91 ± 5.16 weeks. The longest duration of treatment with a uterine contraction inhibitor or inhibitors in combination or with magnesium sulphate alone was 15.34 ± 13.16 days, and nine cases developed adverse reactions. Persistent uterine contraction inhibition after cervical cerclage could prolong pregnancy and improve pregnancy outcomes.Impact statementWhat is already known on this subject? A crucial reason for treatment failure of cervical cerclage is that uterine contraction was not effectively inhibited.What do the results of this study add? Persistent inhibition of uterine contraction after cervical cerclage prolonged pregnancy duration, increased gestational age at delivery, and improved pregnancy outcomes.What are the implications of these findings for clinical practice and/or further research? This study may provide a clinical basis for prolonging gestational age, preventing late abortion and premature delivery, and improving the survival rate and quality of life of premature infants.

Review of the endocrine organ-like tumor hypothesis of cancer cachexia in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal types of solid tumors, associated with a high prevalence of cachexia (~80%). PDAC-derived cachexia (PDAC-CC) is a systemic disease involving the complex interplay between the tumor and multiple organs. The endocrine organ-like tumor (EOLT) hypothesis may explain the systemic crosstalk underlying the deleterious homeostatic shifts that occur in PDAC-CC. Several studies have reported a markedly heterogeneous collection of cachectic mediators, signaling mechanisms, and metabolic pathways, including exocrine pancreatic insufficiency, hormonal disturbance, pro-inflammatory cytokine storm, digestive and tumor-derived factors, and PDAC progression. The complexities of PDAC-CC necessitate a careful review of recent literature summarizing cachectic mediators, corresponding metabolic functions, and the collateral impacts on wasting organs. The EOLT hypothesis suggests that metabolites, genetic instability, and epigenetic changes (microRNAs) are involved in cachexia development. Both tumors and host tissues can secrete multiple cachectic factors (beyond only inflammatory mediators). Some regulatory molecules, metabolites, and microRNAs are tissue-specific, resulting in insufficient energy production to support tumor/cachexia development. Due to these complexities, changes in a single factor can trigger bi-directional feedback circuits that exacerbate PDAC and result in the development of irreversible cachexia. We provide an integrated review based on 267 papers and 20 clinical trials from PubMed and ClinicalTrials.gov database proposed under the EOLT hypothesis that may provide a fundamental understanding of cachexia development and response to current treatments.

Recent Advances in Glycyrrhiza glabra (Licorice)-Containing Herbs Alleviating Radiotherapy- and Chemotherapy-Induced Adverse Reactions in Cancer Treatment.

Cancers represent a significant cause of morbidity and mortality worldwide. They also impose a large economic burden on patients, their families, and health insurance systems. Notably, cancers and the adverse reactions to their therapeutic options, chemotherapy and radiotherapy, dramatically affect the quality of life of afflicted patients. Therefore, developing approaches to manage chemotherapy- and radiotherapy-induced adverse reactions gained greater attention in recent years. Glycyrrhiza glabra (licorice), a perennial plant that is one of the most frequently used herbs in traditional Chinese medicine, has been heavily investigated in relation to cancer therapy. Licorice/licorice-related regimes, used in combination with chemotherapy, may improve the adverse effects of chemotherapy. However, there is little awareness of licorice-containing herbs alleviating reactions to radiotherapy and chemotherapy, or to other induced adverse reactions in cancer treatment. We aimed to provide a descriptive review, and to emphasize the possibility that licorice-related medicines could be used as an adjuvant regimen with chemotherapy to improve quality of life (QoL) and to reduce side effects, thus, improving compliance with chemotherapy. The experimental method involved searching different databases, including PubMed, the Cochrane Library, and Wang Fang database, as of May 2022, to identify any relevant studies. Despite a lack of high-quality and large-scale randomized controlled trials, we still discovered the potential benefits of licorice-containing herbs from published clinical studies. These studies find that licorice-containing herbs, and their active ingredients, reduce the adverse reactions caused by chemotherapy and radiotherapy, and improve the QoL of patients. This comprehensive review will serve as a cornerstone to encourage more scientists to evaluate and develop effective Traditional Chinese medicine prescriptions to improve the side effects of chemotherapy and radiation therapy.

Functional stability of CFTR depends on tight binding of ATP at its degenerate ATP-binding site.

Opening of the cystic fibrosis transmembrane conductance regulator (CFTR) channel is coupled to the motion of its two nucleotide-binding domains: they form a heterodimer sandwiching two functionally distinct ATP-binding sites (sites 1 and 2). While active ATP hydrolysis in site 2 triggers rapid channel closure, the functional role of stable ATP binding in the catalysis-incompetent (or degenerate) site 1, a feature conserved in many other ATP-binding cassette (ABC) transporter proteins, remains elusive. Here, we found that CFTR loses its prompt responsiveness to ATP after the channel is devoid of ATP for tens to hundreds of seconds. Mutants with weakened ATP binding in site 1 and the most prevalent disease-causing mutation, F508del, are more vulnerable to ATP depletion. In contrast, strengthening ligand binding in site 1 with N6 -(2-phenylethyl)-ATP, a high-affinity ATP analogue, or abolishing ATP hydrolysis in site 2 by the mutation D1370N, helps sustain a durable function of the otherwise unstable mutant channels. Thus, tight binding of ATP in the degenerate ATP-binding site is crucial to the functional stability of CFTR. Small molecules targeting site 1 may bear therapeutic potential to overcome the membrane instability of F508del-CFTR. KEY POINTS: During evolution, many ATP-binding cassette transporters - including the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, whose dysfunction causes cystic fibrosis (CF) - lose the ability to hydrolyse ATP in one of the two ATP-binding sites. Here we show that tight ATP binding at this degenerate site in CFTR is central for maintaining the stable, robust function of normal CFTR. We also demonstrate that membrane instability of the most common CF-causing mutant, F508del-CFTR, can be rescued by strengthening ATP binding at CFTR's degenerate site. Our data thus explain an evolutionary puzzle and offer a potential therapeutic strategy for CF.

Perspectives on the Role of Isoliquiritigenin in Cancer.

Isoliquiritigenin (2',4',4-trihydroxychalcone, ISL), one of the most important bioactive compounds with a chalcone structure, is derived from licorice root. Licorice is commonly known as Glycyrrhiza, including Glycyrrhiza uralensis, Glycyrrhiza radix, and Glycyrrhiza glabra, which are generally available in common foods and Chinese herbal medicines based on a wide variety of biological functions and pharmacological effects, and its derivative (ISL) is utilized as a food additive and adjunct disease treatment. In this review, we summarized the progress over the last 10 years in the targeted pathways and molecular mechanisms of ISL that are involved in the regulation of the onset and progression of different types of cancers.

Structural mechanisms for defective CFTR gating caused by the Q1412X mutation, a severe Class VI pathogenic mutation in cystic fibrosis.

KEY POINTS: Electrophysiological characterization of Q1412X-CFTR, a C-terminal truncation mutation of cystic fibrosis transmembrane conductance regulator (CFTR) associated with the severe form of cystic fibrosis (CF), reveals a gating defect that has not been reported previously. Mechanistic investigations of the gating deficit in Q1412X-CFTR suggest that the reduced open probability in Q1412X-CFTR is the result of a disruption of the function of the second ATP binding site (or site 2) in the nucleotide binding domains (NBDs). Detailed comparisons of several mutations with different degrees of truncation in the C-terminal region of NBD2 reveal the importance of the last two beta-strands in NBD2 for maintaining proper gating functions. The results of the present study also show that the application of clinically-approved drugs (VX-770 and VX-809) can greatly enhance the function of Q1412X, providing in vitro evidence for a therapeutic strategy employing both reagents for patients bearing Q1412X or similar truncation mutations. ABSTRACT: Cystic fibrosis (CF) is caused by loss-of-function mutations of cystic fibrosis transmembrane conductance regulator (CFTR), a phosphorylation-activated but ATP-gated chloride channel. Based on the molecular mechanism of CF pathogenesis, disease-associated mutations are categorized into six classes. Among them, Class VI, whose members include some of the C-terminal truncation mutations such as Q1412X, is defined as decreased membrane expression because of a faster turnover rate. In the present study, we characterized the functional properties of Q1412X-CFTR, a severe-form premature stop codon mutation. We confirmed previous findings of a ∼90% decrease in membrane expression but found a ∼95% reduction in the open probability (Po ). Detailed kinetic studies support the idea that the gating defect is the result of a dysfunctional ATP-binding site 2 in the nucleotide binding domains (NBDs). Because the Q1412X mutation results in a deletion of the last two beta-strands in NBD2 and the whole C-terminal region, we further characterized truncation mutations with different degrees of deletion in this segment. Mutations that completely or partially remove the C-terminus of CFTR at the same time as keeping an intact NBD2 (i.e. D1425X and S1455X) assume gating function almost identical to that of wild-type channels. However, the deletion of the last beta-strand in the NBD2 (i.e. N1419X) causes gating dysfunction that is milder than that of Q1412X. Thus, normal CFTR gating requires structural integrity of NBD2. Moreover, our observation that clinically-approved VX-809 (Lumacaftor, Vertex Pharmaceuticals, Boston, MA, USA) and VX-770 (Ivacaftor, Vertex Pharmaceuticals, Boston, MA, USA) significantly enhance the overall function of Q1412X-CFTR provides the conceptual basis for the treatment of patients carrying this mutation.

Structural mechanisms of CFTR function and dysfunction.

Cystic fibrosis (CF) transmembrane conductance regulator (CFTR) chloride channel plays a critical role in regulating transepithelial movement of water and electrolyte in exocrine tissues. Malfunction of the channel because of mutations of the cftr gene results in CF, the most prevalent lethal genetic disease among Caucasians. Recently, the publication of atomic structures of CFTR in two distinct conformations provides, for the first time, a clear overview of the protein. However, given the highly dynamic nature of the interactions among CFTR's various domains, better understanding of the functional significance of these structures requires an integration of these new structural insights with previously established biochemical/biophysical studies, which is the goal of this review.

Functional characterization reveals that zebrafish CFTR prefers to occupy closed channel conformations.

Cystic fibrosis transmembrane conductance regulator (CFTR), the culprit behind the genetic disease cystic fibrosis (CF), is a phosphorylation-activated, but ATP-gated anion channel. Studies of human CFTR over the past two decades have provided an in-depth understanding of how CFTR works as an ion channel despite its structural resemblance to ABC transporters. Recently-solved cryo-EM structures of unphosphorylated human and zebrafish CFTR (hCFTR and zCFTR), as well as phosphorylated ATP-bound zebrafish and human CFTR offer an unprecedented opportunity to understand CFTR's function at a molecular level. Interestingly, despite millions of years of phylogenetic distance between human and zebrafish, the structures of zCFTR and hCFTR exhibit remarkable similarities. In the current study, we characterized biophysical and pharmacological properties of zCFTR with the patch-clamp technique, and showed surprisingly very different functional properties between these two orthologs. First, while hCFTR has a single-channel conductance of 8.4 pS with a linear I-V curve, zCFTR shows an inwardly-rectified I-V relationship with a single-channel conductance of ~3.5 pS. Second, single-channel gating behaviors of phosphorylated zCFTR are very different from those of hCFTR, featuring a very low open probability Po (0.03 ± 0.02, vs. ~0.50 for hCFTR) with exceedingly long closed events and brief openings. In addition, unlike hCFTR where each open burst is clearly defined with rare short-lived flickery closures, the open bursts of zCFTR are not easily resolved. Third, although abolishing ATP hydrolysis by replacing the catalytic glutamate with glutamine (i.e., E1372Q) drastically prolongs the open bursts defined by the macroscopic relaxation analysis in zCFTR, the Po within a "locked-open" burst of E1372Q-zCFTR is only ~ 0.35 (vs. Po > 0.94 in E1371Q-hCFTR). Collectively, our data not only provide a reasonable explanation for the unexpected closed-state structure of phosphorylated E1372Q-zCFTR with a canonical ATP-bound dimer of the nucleotide binding domains (NBDs), but also implicate significant structural and functional differences between these two evolutionarily distant orthologs.

On the mechanism of gating defects caused by the R117H mutation in cystic fibrosis transmembrane conductance regulator.

KEY POINTS: Two functional abnormalities of cystic fibrosis transmembrane conductance regulator (CFTR), a 25% reduction of the single-channel conductance (g) and a ∼13-fold lower open probability (Po ), were found with the R117H mutation that is associated with mild forms of cystic fibrosis. Characterizations of the gating defects of R117H-CFTR led to the conclusion that the mutation decreases Po by perturbing the gating conformational changes in CFTR's transmembrane domains (TMDs) without altering the function of the nucleotide binding domains (NBDs). Nonetheless, gating of the R117H-CFTR can be improved by a variety of pharmacological reagents supposedly acting on NBDs such as ATP analogues, or TMDs (e.g. VX-770 or nitrate). These reagents potentiate synergistically R117H-CFTR gating to a level that allows accurate assessments of its gating deficits. Our studies not only elucidate the mechanism underpinning gating dysfunction of R117H-CFTR, but also provide a mechanistic understanding of how VX-770 ameliorates the gating defects in the R117H mutant. ABSTRACT: Cystic fibrosis (CF) is caused by loss-of-function mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene encoding a phosphorylation-activated, but ATP-gated chloride channel. In the current study, we investigated the mechanism responsible for the gating defects manifested in R117H-CFTR, an arginine-to-histidine substitution at position 117 of CFTR that is associated with mild forms of CF. We confirmed previous findings of a 25% decrease of the single-channel conductance (g) in R117H-CFTR, but found a ∼13-fold lower open probability (Po ). This dramatic gating deficit is not due to dysfunctional nucleotide binding domains (NBDs) as the mutation does not alter the apparent affinity for ATP, and the mutant channels respond to ATP analogues in a similar manner as wild-type CFTR. Furthermore, once ATP hydrolysis is abolished, the R117H mutant can be trapped in a prolonged 'burst opening' conformation that is proposed to be equipped with a stable NBD dimer. On the other hand, our results support the conclusion that the R117H mutation decreases Po by perturbing the gating conformational changes in CFTR's transmembrane domains as even when NBDs are kept at a dimerized configuration, Po is reduced by ∼10-fold. Moreover, our data demonstrate that a synergistic improvement of R117H-CFTR function can be accomplished with a combined regiment of VX-770 (Ivacaftor), nitrate ion (NO3 (-) ) and N(6) -(2-phenylethyl)-2'-deoxy-ATP (d-PATP), which almost completely rectifies the gating defect of R117H-CFTR. Clinical implications of our results are discussed.

Direct visualization and quantitative analysis of water diffusion in complex biological tissues using CARS microscopy.

To date, it has not been possible to measure microscopic diffusive water movements in epithelia and in the interstitial space of complex tissues and organs. Diffusive water movements are essential for life because they convey physiologically important small molecules, e.g. nutrients and signaling ligands throughout the extracellular space of complex tissues. Here we report the development of a novel method for the direct observation and quantitative analysis of water diffusion dynamics in a biologically organized tissue using Coherent Anti-Stokes Raman Scattering (CARS) microscopy. Using a computer simulation model to analyze the CARS O-H bond vibration data during H2O/D2O exchange in a 3D epithelial cyst, we succeeded in measuring the diffusive water permeability of the individual luminal and basolateral water pathways and also their response to hormonal stimulation. Our technique will be applicable to the measurement of diffusive water movements in other structurally complex and medically important tissues and organs.

Curcumin and genistein: the combined effects on disease-associated CFTR mutants and their clinical implications.

Genistein and curcumin are major components of Asian foods, soybean and curry turmeric respectively. These compounds have been intensively investigated for their chemical and biological features conferring their anti-cancer activity. Genistein and curcumin have also been investigated for their potentiation effects on disease-associated CFTR mutants such as ΔF508 and G551D. Recently, we investigated the combined effect of genistein and curcumin on G551D-CFTR, which exhibits gating defects without abnormalities in protein synthesis or trafficking using the patch-clamp technique. We found that genistein and curcumin showed additive effects on their potentiation of G551D-CFTR in high concentration range and also, more importantly, showed a significant synergistic effect in their minimum concentration ranges. These results are consistent with the idea that multiple mechanisms are involved in the action of these CFTR potentiators. In this review, we revisit the pharmacology of genistein and curcumin on CFTR and also propose new pharmaceutical implications of combined use of these compounds in the development of drugs for CF pharmacotherapy.

Curcumin and genistein additively potentiate G551D-CFTR.

BACKGROUND: The G551D mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) is a common cause of cystic fibrosis (CF). G551D-CFTR is characterized by an extremely low open probability despite its normal trafficking to the plasma membrane. Numerous small molecules have been shown to increase the activity of G551D-CFTR presumably by binding to the CFTR protein. METHODS: We investigated the effect of curcumin, genistein and their combined application on G551D-CFTR activity using the patch clamp technique. RESULTS: Curcumin increased G551D-CFTR whole-cell and single-channel currents less than genistein did at their maximally effective concentrations. However, curcumin further increased the channel activity of G551D-CFTR that had been already maximally potentiated by genistein, up to ~50% of the WT-CFTR level. In addition, the combined application of genistein and curcumin over a lower concentration range synergistically rescued the gating defect of G551D-CFTR. CONCLUSIONS: The additive effects between curcumin and genistein not only support the hypothesis that multiple mechanisms are involved in the action of CFTR potentiators, but also pose pharmaceutical implications in the development of drugs for CF pharmacotherapy.

A stable ATP binding to the nucleotide binding domain is important for reliable gating cycle in an ABC transporter CFTR.

Cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, a member of ABC transporter superfamily, gates following ATP-dependent conformational changes of the nucleotide binding domains (NBD). Reflecting the hundreds of milliseconds duration of the channel open state corresponding to the dimerization of two NBDs, macroscopic WT-CFTR currents usually showed a fast, single exponential relaxation upon removal of cytoplasmic ATP. Mutations of tyrosine1219, a residue critical for ATP binding in second NBD (NBD2), induced a significant slow phase in the current relaxation, suggesting that weakening ATP binding affinity at NBD2 increases the probability of the stable open state. The slow phase was effectively diminished by a higher affinity ATP analogue. These data suggest that a stable binding of ATP to NBD2 is required for normal CFTR gating cycle, andthat the instability of ATP binding frequently halts the gating cycle in the open state presumably through a failure of ATP hydrolysis at NBD2.

[Construction, expression and purification of repeat fragment polymer of oInhibin alpha-subunit N terminal 1-33 fragment].

A cDNA sequence coding for ovine inhibin N terminal 1-33 AA residue fragment (INH) was inserted between BamHI\SacI sites in plasmid pRSET-A to generate plasmid pR-INH. By utilizing a pair of isocaudamer BamHI and Bgl II sites and another downstream Hind III site, following simple double digestions and combination ligation of the resultant products, 2 to 6-repeat INH genes were constructed respectively. Each plamids containing 3 to 6 repeated INH fragment genes, pR-3INH, pR-4INH, pR-5INH and pR-6INH, directed expression of the target proteins in E. coli. BL21 (DE3) under induction of ITPG, which respectively accounted for 6%, 6%, 7% and 8% of the total bacterial protein. The expressed target proteins were all in the form of inclusion bodies. The above results implied that utilization of isocaudamer restriction disgetion sites in expression plasmid is capable of rapidly and correctly constructing repeat fragment polymer of short peptides, which may become a new method in construction of high immunogenic recombinant vaccines of short peptides.