Modulation of caspase activation and p27(Kip1) degradation in the p53-induced apoptosis in IW32 erythroleukemia cells

To examine the p53-mediated biological activities and signalling pathways, we generated stable transfectants of the p53-null IW32 murine erythroleukemia cells expressing the temperature-sensitive p53 mutant DNA, tsp53(val135). Two clones with different levels of p53 protein expression were selected for further characterization. At permissive temperature, clone 1-5 cells differentiated along the erythroid pathway, and clone 3-2 cells that produced greater levels (3.5-fold) of p53 underwent apoptosis. Apoptosis of 3-2 cells was accompanied by mitochondrial cytochrome c release and caspase activation as well as by cleavage of caspase substrates. Bax protein was induced to a similar extent in these clones by wild-type p53; expression of p21(Cip1/Waf1) and p27(Kip1) proteins was also increased. However, significantly lesser extent of induction for both CDK inhibitors was detected in the apoptotic 3-2 clone. The general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD.fmk) blocked the p53-induced apoptosis in 3-2 cells, with a concomitant elevation of p27(Kip1), suggesting that p27(Kip1) protein underwent caspase-dependent proteolysis in the apoptotic 3-2 cells. Together these results linked a pathway involving cytochrome c release, caspase activation and p27(Kip1) degradation to the p53-induced apoptosis in IW32 erythroleukemia cells.     

C-Terminal Helical Domains of Dengue Virus Type 4 E Protein Affect the Expression/Stability of prM Protein and Conformation of prM and E Proteins

Background

The envelope (E) protein of dengue virus (DENV) is the major immunogen for dengue vaccine development. At the C-terminus are two α-helices (EH1 and EH2) and two transmembrane domains (ET1 and ET2). After synthesis, E protein forms a heterodimer with the precursor membrane (prM) protein, which has been shown as a chaperone for E protein and could prevent premature fusion of E protein during maturation. Recent reports of enhancement of DENV infectivity by anti-prM monoclonal antibodies (mAbs) suggest the presence of prM protein in dengue vaccine is potentially harmful. A better understanding of prM-E interaction and its effect on recognition of E and prM proteins by different antibodies would provide important information for future design of safe and effective subunit dengue vaccines.

Methodology/Principal Findings

In this study, we examined a series of C-terminal truncation constructs of DENV4 prME, E and prM. In the absence of E protein, prM protein expressed poorly. In the presence of E protein, the expression of prM protein increased in a dose-dependent manner. Radioimmunoprecipitation, sucrose gradient sedimentation and pulse-chase experiments revealed ET1 and EH2 were involved in prM-E interaction and EH2 in maintaining the stability of prM protein. Dot blot assay revealed E protein affected the recognition of prM protein by an anti-prM mAb; truncation of EH2 or EH1 affected the recognition of E protein by several anti-E mAbs, which was further verified by capture ELISA. The E protein ectodomain alone can be recognized well by all anti-E mAbs tested.

Conclusions/Significance

A C-terminal domain (EH2) of DENV E protein can affect the expression and stability of its chaperone prM protein. These findings not only add to our understanding of the interaction between prM and E proteins, but also suggest the ectodomain of E protein alone could be a potential subunit immunogen without inducing anti-prM response.     

Pancreatic B-cell function in non-insulin-dependent diabetes mellitus during successive periods of sulfonylurea and insulin treatment: serum C-peptide response to glucagon and urine C-peptide excretion

Serum C-peptide responses to glucagon and daily urine C-peptide excretion in successive periods of different treatment in two groups of patients with non-insulin-dependent diabetes mellitus (NIDDM) (mean interval between two tests less than 1 month) were compared. In group A patients (n = 8), the glycemic control was improved after transferring the treatment from sulfonylurea (SU) to insulin (fasting plasma glucose: SU: 192 +/- 47, insulin: 127 +/- 21 mg/dl, mean +/- S.D., p less than 0.01). Fasting serum C-peptide immunoreactivity (CPR) was significantly lower at the period of insulin treatment (SU: 1.93 +/- 1.01, insulin: 1.47 +/- 0.79 ng/ml, p less than 0.05), but there was no difference in the increase in serum CPR (maximal--fasting) (delta serum CPR) during glucagon stimulation in the two periods of treatment (SU: 1.70 +/- 0.72, insulin: 1.47 +/- 0.98 ng/ml). In group B patients (n = 7), there was no significant difference in glycemic control after transferring the treatment from insulin to SU (fasting plasma glucose: insulin: 127 +/- 24, SU: 103 +/- 13 mg/dl). Fasting serum CPR was significantly lower during the period of insulin treatment (insulin: 1.39 +/- 0.64, SU: 2.21 +/- 0.86 ng/ml, p less than 0.025), but delta serum CPR during glucagon stimulation still showed no significant difference between the two periods (insulin: 1.97 +/- 1.16, SU: 2.33 +/- 1.57 ng/ml).(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequence dependence of Drosophila topoisomerase II in plasmid relaxation and DNA binding

The sequence dependence of Drosophila topoisomerase II supercoil relaxation and binding activities has been examined. The DNA substrates used in binding experiments were two fragments from Drosophila heat shock locus 87A7. One of these DNA fragments includes the coding region for the heat shock protein hsp70, and the other includes the intergenic non-coding region that separates two divergently transcribed copies of the hsp70 gene at the locus. The intergenic region was previously shown to have a much higher density of topoisomerase cleavage sites than the hsp70 coding region. Competition nitrocellulose filter binding assays demonstrate a preferential binding of the intergene fragment, and that binding specificity increases with increasing ionic strength. Dissociation kinetics indicate a greater kinetic stability of topoisomerase II complexes with the intergene DNA fragment. To study topoisomerase II relaxation activity, we used supercoiled plasmids that contained the same fragments from locus 87A7 cloned as inserts. The relative relaxation rates of the two plasmids were determined under several conditions of ionic strength, and when the plasmid substrates were included in separate reactions or when they were mixed in a single reaction. The relaxation properties of these two plasmids can be explained by a coincidence of high-affinity binding sites, strong cleavage sites, and sites used during the catalysis of strand passage events by topoisomerase II. Sequence dependence of topoisomerase II catalytic activity may therefore parallel the sequence dependence of DNA cleavage by this enzyme.     

Antifungal properties of ethanolic extract and its active compounds from Calocedrus macrolepis var. formosana (Florin) heartwood

The ethanolic extract of Calocedrus macrolepis var. formosana heartwood was screened for antifungal compounds by agar dilution assay and liquid chromatography. Two compounds, beta-thujaplicin and gamma-thujaplicin, responsible for the antifungal property of C. macrolepis var. formosana heartwood were isolated by high performance liquid chromatography (HPLC), and identified by 1H NMR and 13C NMR. The antifungal activities of these two compounds were further evaluated against total 15 fungi, including wood decay fungi, tree pathogenic fungi and molds. The hexane soluble fraction showed the strongest antifungal activities among all fractions. beta-Thujaplicin and gamma-thujaplicin exhibited not only very strong antifungal activity, but also broad antifungal spectrum. The MIC values of beta-thujaplicin and gamma-thujaplicin were in the range of 5.0-50.0 microg/ml. In addition, scanning electron microscopy (SEM) was carried out to study the structural change of fungal hyphae induced by beta-thujaplicin. Strong cell wall shrinkage indicated the fungicidal effect could be attributed to the combined actions of metal chelating and cytoplasm leakage. It also suggests that the role of metal chelating is indispensable in the design of environmental-friendly fungicides.     

Aliskiren Attenuates Steatohepatitis and Increases Turnover of Hepatic Fat in Mice Fed with a Methionine and Choline Deficient Diet

Background & Aims

Activation of the renin-angiotensin-system is known to play a role in nonalcoholic steatohepatitis. Renin knockout mice manifest decreased hepatic steatosis. Aliskiren is the first direct renin inhibitor to be approved for clinical use. Our study aims to evaluate the possible therapeutic effects and mechanism of the chronic administration of aliskiren in a dietary steatohepatitis murine model.

Methods

Male C57BL/6 mice were fed with a methionine and choline-deficient (MCD) diet to induce steatohepatitis. After 8 weeks of feeding, the injured mice were randomly assigned to receive aliskiren (50 mg_·kg^-1 per day) or vehicle administration for 4 weeks. Normal controls were also administered aliskiren (50 mg_·kg^-1 per day) or a vehicle for 4 weeks.

Results

In the MCD mice, aliskiren attenuated hepatic steatosis, inflammation and fibrosis. Aliskiren did not change expression of lipogenic genes but increase turnover of hepatic fat by up-regulating peroxisome proliferator-activated receptor α, carnitine palmitoyltransferase 1a, cytochrome P450-4A14 and phosphorylated AMP-activated protein kinase. Furthermore, aliskiren decreased the hepatic expression of angiotensin II and nuclear factor κB. The levels of oxidative stress, hepatocyte apoptosis, activation of Kupffer cells and hepatic stellate cells, and pro-fibrotic markers were also reduced in the livers of the MCD mice receiving aliskiren.

Conclusions

Aliskiren attenuates steatohepatitis and fibrosis in mice fed with a MCD diet. Thus, the noted therapeutic effects might come from not only the reduction of angiotensin II but also the up-regulation of fatty acid oxidation-related genes.     

Studies on somaclonal variation in Phalaenopsis

The morphological and genetic variations in somaclones of Phalaenopsis True Lady “B79-19” derived from tissue culture were evaluated. In 1360 flowering somaclones, no apparent difference was found in the shape of the leaves, whereas flowers in some somaclones were deformed. We have demonstrated that 38 selected random primers can be used to generate amplified segments of genomic DNA and to differentiate polymorphisms of somaclonal variations in Phalaenopsis. The random amplified polymorphic DNA (RAPD) data indicated that normal and variant somaclones are not genetically identical. We also studied the banding patterns of aspartate aminotransferase (AAT) and phosphoglucomutase (PGM) in young leaves of variant and normal somaclones of Phalaenopsis. With respect to AAT, three distinct banding patterns were found in normal somaclones and only two-banded phenotypes were detected in variant somaclones. In a comparison of the banding patterns of PGM isozymes, three to four bands were detected in normal somaclones and two to three bands in variant ones. Received: 15 August 1997 / Revision received: 16 February 1998 / Accepted: 1 May 1998     

Requirement for Phe36 for DNA binding and mismatch repair by Escherichia coli MutS protein

The MutS family of DNA repair proteins recognizes base pair mismatches and insertion/deletion mismatches and targets them for repair in a strand-specific manner. Photocrosslinking and mutational studies previously identified a highly conserved Phe residue at the N-terminus of Thermus aquaticus MutS protein that is critical for mismatch recognition in vitro. Here, a mutant Escherichia coli MutS protein harboring a substitution of Ala for the corresponding Phe36 residue is assessed for proficiency in mismatch repair in vivo and DNA binding and ATP hydrolysis in vitro. The F36A protein is unable to restore mismatch repair proficiency to a mutS strain as judged by mutation to rifampicin or reversion of a specific point mutation in lacZ. The F36A protein is also severely deficient for binding to heteroduplexes containing an unpaired thymidine or a G:T mismatch although its intrinsic ATPase activity and subunit oligomerization are very similar to that of the wild-type MutS protein. Thus, the F36A mutation appears to confer a defect specific for recognition of insertion/deletion and base pair mismatches.     

IL-31 Associated with Coronary Artery Lesion Formation in Kawasaki Disease

Background

Kawasaki disease (KD) is known to be associated with T help (Th) 2 reaction and subsequently allergic diseases. Interleukin-31 (IL-31) has also been reported to be involved in Th2 mediated diseases such as allergic diseases. However, the role of IL-31 in KD has not been previously reported. The aim of this study is to investigate whether IL-31 is associated with KD and its clinical outcome.

Material

A total of 78 KD patients who met the criteria of KD were enrolled in this study as well as 20 age-matched controls. Plasma samples were conducted to measure IL-31 before intravenous immunoglobulin (IVIG) treatment (KD1), within 3 days after IVIG treatment (KD2) and at least 3 weeks after IVIG treatment (KD3) by utilizing enzyme-linked immunosorbent assay (ELISA).

Result

Our findings showed that IL-31 expression was higher in KD patients after IVIG treatment significantly (KD2>KD1: 1265.0±199.3 vs. 840.2±152.5 pg/ml, p<0.0001). Further analysis revealed that IL-31 level was significantly higher in KD patients with coronary artery lesion (CAL) (656.6±139.5 vs. 1373.0±422.0 pg/ml, p = 0.04) before IVIG treatment (KD1). There were no significant differences between the IVIG resistance and IVIG responsiveness groups.

Conclusion

IL-31 was increased after IVIG treatment in patients with KD and was significantly associated with CAL formation. The results from this study may help to identify a novel risk factor for predicting KD and CAL formation.