Betulinic acid inhibits high-fat diet-induced obesity and improves energy balance by activating AMPK.

BACKGROUND AND AIM: Metabolic syndromes are prevalent worldwide and result in various complications including obesity, cardiovascular disease and type II diabetes. Betulinic acid (BA) is a naturally occurring triterpenoid that has anti-inflammatory properties. We hypothesized that treatment with BA may result in decreased body weight gain, adiposity and hepatic steatosis in a diet-induced mouse model of obesity. METHODS AND RESULTS: Mice fed a high-fat diet and treated with BA showed less weight gain and tissue adiposity without any change in calorie intake. Gene expression profiling of mouse tissues and cell lines revealed that BA treatment increased expression of lipid oxidative genes and decreased that of lipogenesis-related genes. This modulation was mediated by increased AMP-activated protein kinase (AMPK) phosphorylation, which facilitates energy expenditure, lipid oxidation and thermogenic capacity and exerts protective effects against obesity and nonalcoholic fatty liver disease. Overall, BA markedly inhibited the development of obesity and nonalcoholic fatty liver disease in mice fed a high-fat diet, and AMPK activation in various tissues and enhanced thermogenesis are two possible mechanisms underlying the antiobesity and antisteatogenic effects of BA. CONCLUSIONS: The current findings suggest that treatment with BA is a potential dietary strategy for preventing obesity and nonalcoholic fatty liver disease.

Utilization of algal sugars and glycerol for enhanced cephalosporin C production by Acremonium chrysogenum M35.

In our previous study, glycerol was utilized as an additional carbon source for the production of cephalosporin C (CPC) by Acremonium chrysogenum M35. In this study, algal sugars extracted from the third-generation biomass were utilized in the CPC production for the first time. The CPC production improved about twofold when using the algal sugars as the carbon source. The complex medium including algal sugars and glycerol was utilized, and 7·3 g l-1 CPC production was achieved in a 250-ml shaking flask. To determine the important variables for the CPC production, Plackett-Burman design was carried out and 6·18 g l-1 of CPC was estimated under the numerically optimized conditions. Under the optimized conditions, the CPC production was performed in a 5-l scale bioreactor, affording CPC production at a rate of 7·1 g l-1 . Moreover, 6·7 g l-1 CPC was produced using crude glycerol as the substrate. SIGNIFICANCE AND IMPACT OF THE STUDY: Microalgae are the biomass containing various components, such as carbohydrates, lipids, and amino acids. In this study, carbon sources contained in microalgae were obtained by acid extraction, and cephalosporin C (CPC), a ß-lactam antibiotic intermediate, was produced by using Acremonium chrysogenum M35. In addition, the increase of CPC production was not distinct for A. chrysogenum M35 with algal sugars as the only carbon source; therefore, glycerol was added, increasing the CPC production. Thus, cheap residues such as algal sugars form microalgal and glycerol form biodiesel process could be used as the alternative sources for the production of various products.

A phase II study of everolimus (RAD001), an mTOR inhibitor plus CHOP for newly diagnosed peripheral T-cell lymphomas.

BACKGROUND: Everolimus, an oral mTOR inhibitor, has single-agent activity against relapsed lymphomas. Thus, we carried out a phase II study of everolimus in combination with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) as a first-line treatment for patients with peripheral T-cell lymphoma (PTCL) based on our phase I study results. PATIENTS AND METHODS: Participants (n = 30) received CHOP with 5 mg everolimus per day from day 1 to 14 every 21 days for a total of six cycles. The primary end point was the overall response rate (ORR), which included complete response (CR) and partial response (PR) to this regimen. Immunohistochemistry was used to evaluate the expression of phosphatase and tensin homology (PTEN) and phosphorylated S6 kinase (pS6K) as a response. RESULTS: The objective response rate was 90% with CR (n = 17) and PR (n = 10). The CR rate was different among subtypes; angioimmunoblastic T-cell lymphoma (AITL, n = 3) had a CR whereas PTCL-not-otherwise specified and ALK-negative anaplastic large-cell lymphoma (ALCL) patients showed 63% (12/19) and 29% (2/7) of CR rate, respectively. This difference in CR rate among subtypes was associated with PTEN loss because PTEN loss was not seen in AITL but 33% of ALCL patients. The most common toxicity was hematological, with 80% of patients experiencing at least one event of grade 3/4 neutropenia, and 60% of patients had grade 3/4 thrombocytopenia. CONCLUSION: The everolimus plus CHOP was effective for PTCL patients, and its efficacy might be related with the preservation of PTEN.

Role of arachidonic acid-derived metabolites in the control of pulmonary arterial pressure and hypoxic pulmonary vasoconstriction in rats.

BACKGROUND: The roles of arachidonic acid (AA) metabolites in hypoxia-induced pulmonary vasoconstriction (HPV), a critical physiological mechanism that prevents ventilation/perfusion mismatch, are still incompletely understood. METHODS: Pulmonary arterial pressure was measured in ventilated/perfused rat lungs. Isometric tones of rat intralobar pulmonary arteries were also measured, using a myograph. RESULTS: Hypoxia (Po2, 3%)-induced pulmonary arterial pressure increases (ΔPAP(hypox)) were stable with blood-mixed perfusate, but decayed spontaneously. ΔPAP(hypox) was inhibited by 29%, 16%, and 28% by the thromboxane A2 (TXA2) antagonist SQ-29548, the 5-lipoxygenase inhibitor, MK886, and the leukotriene D4 antagonist, LY-171883, respectively. The prostacyclin synthase inhibitor tranylcypromine augmented ΔPAP(hypox) by 5%, whereas inhibition of cytochrome P450 did not affect ΔPAP(hypox). Consistently, the TXA2 analogue U46619 increased ΔPAP(hypox) whereas prostacyclin abolished ΔPAP(hypox). However, leukotriene D4 had no direct effect on ΔPAP(hypox). In the isolated pulmonary arteries, pretreatment with U46619 was essential to demonstrate hypoxia-induced contraction. CONCLUSIONS: The above results suggest that TXA2 and cysteinyl leukotrienes, other than leukotriene D4, are endogenous factors that facilitate HPV in rats. The indispensable role of TXA2-induced pretone in the HPV of isolated pulmonary arteries indicates that the signal from thromboxane receptors might be a critical component of oxygen sensation mechanisms.

Effects of sevoflurane on the cAMP-induced short-circuit current in mouse tracheal epithelium and recombinant Cl- (CFTR) and K+ (KCNQ1) channels.

BACKGROUND: An optimal level of airway surface liquid is essential for mucociliary clearance in lungs. The cAMP-activated cystic fibrosis transmembrane conductance regulator (CFTR) and KCNQ1 channels in tracheal epithelium play key roles in luminal and basolateral membranes, respectively. The aim of this study was to examine the effects of sevoflurane on cAMP-induced chloride secretion by the mouse tracheal epithelium and the modulation of recombinant CFTR and KCNQ1 channels. METHODS: The equivalent short-circuit current (Isc) of the mouse tracheal epithelium was measured using a flow-type Ussing chamber technique. Inhibition of Na+ absorption was achieved through the luminal application of amiloride. cAMP-dependent Cl- secretion was evoked by forskolin and isobutylmethylxanthine (Fsk/IBMX) applied to the basolateral side. The effect of sevoflurane on CFTR and KCNQ1 channels was assessed using a whole-cell patch clamp in human embryonic kidney 293T cells expressing CFTR and KCNQ1 channels. RESULTS: Fsk/IBMX induced a sustained Isc that was suppressed by the application of sevoflurane [decreased by 49 (4.5)% at 190 microM]. The Fsk/IBMX-induced Isc was also blocked by basolateral application of chromanol 293B, a blocker of the KCNQ1 K+ channel. In KCNQ1-expressing cells, sevoflurane 190 microM reduced the outward currents to 59 (4.9)% at 80 mV. The CFTR current was not affected by sevoflurane (approximately 360 microM). CONCLUSIONS: These results suggest that the inhibition of KCNQ1 underlies sevoflurane-induced decrease in airway secretion.

Paraduodenal hernia: a treatable cause of upper gastrointestinal tract symptoms.

Paraduodenal hernia (PDH) is an unusual condition that is caused by congenital intestinal malrotation. Noncatastrophic presenting symptoms and their responses to surgery have not been well-characterized. Barium upper gastrointestinal (UGI) series and small bowel follow-up x-rays, performed from December 1995 to September 1996, were sequentially reviewed by one radiologist (J.M.) to identify patients with small bowel series compatible with a PDH. Case histories were reviewed for symptomatic presentation, associated evaluation, and treatment. Based on the 294 UGIs and small bowel follow-throughs performed during this 10-month period, 6 cases were suspected to have a PDH. A right PDH was confirmed in the three patients who underwent surgical exploration (prevalence 1%). Preoperative patient symptoms included nausea, bilious vomiting, and right upper quadrant pain. Repair of the hernia defect resulted in complete resolution of chronic symptoms. Preoperative upper endoscopy, performed in three patients, was not helpful in identifying the disorder. Preoperative computerized tomography obtained in two patients was diagnostic for a right PDH. One symptomatic patient with vomiting and gastric stasis did not have surgery because of a terminal illness. The remaining two patients had no symptoms attributable to PDH. Patients with PDH frequently have chronic UGI symptoms. An upper endoscopy cannot be used to exclude this entity. After surgery, UGI symptoms from PDH are likely to resolve.

Selective transcriptional regulations in the human liver cell by hepatitis B viral X protein.

The hepatitis B viral X protein (HBx) is known as a transcription factor and potential oncogene. To gain a better view of the effect of HBx on the transcriptional regulation in the human liver cell, we constructed a HepG2 cell line stably expressing HBx (HepG2-HBx), and performed cDNA microarray analysis on 588 cellular cDNAs comparing with untransformed control cells. Two genes (IGFR-2, RhoA) of oncogenes, one gene (p55CDC) of cell cycle regulators, three genes (thrombin receptor, MLK-3, MacMARCKS) of intracellular transducers, one gene (HSP27) of stress response proteins, two genes (FAST kinase, Bak) of apoptosis response proteins, one gene (p21(WAF)) of transcription factors were highly up-regulated; one gene (transcription elongation factor SII) of transcription factors and two genes (monocyte chemotactic protein 1, T-lymphocyte-secreted protein I-309) of growth factors were highly down-regulated. These results showed selective transcriptional regulation by HBx in the human liver cell.

Heavy metal-mediated activation of the rat Cu/Zn superoxide dismutase gene via a metal-responsive element.

The Cu/Zn superoxide dismutase (SODI) catalyzes the dismutation of superoxide radicals produced in the course of biological oxidations. When placed under the control of the rat SOD1 gene promoter and transfected into human HepG2 hepatoma cells, the activity of a chloramphenicol acetyltransferase reporter gene was found to increase three- to four-fold in the presence of heavy metals (cadmium, zinc and copper). Functional analysis of mutant derivatives of the SOD1 gene promoter and the use of a heterologous promoter system confirmed that the induction of the SOD1 gene by metal ions requires a metal-responsive element (MRE) located between positions -273 and -267 (GCGCGCA). It was also shown by gel mobility shift assays that an MRE binding protein is induced by the exposure of the human liver cell line HepG2 to heavy metals. These results suggest that the MRE participates in the induction of the SOD1 gene by heavy metals.

The activation of the rat copper/zinc superoxide dismutase gene by hydrogen peroxide through the hydrogen peroxide-responsive element and by paraquat and heat shock through the same heat shock element.

Copper/zinc superoxide dismutase (SOD1) protects cells against oxidative hazards by the dismutation of superoxide radicals. The promoter activity of the SOD1 gene was increased 3-5-fold by hydrogen peroxide, paraquat (PQ) and heat shock. Functional analyses of the regulatory region of the SOD1 gene by deletions, mutations, and heterologous promoter systems confirmed the induction of the SOD1 gene by H(2)O(2) through the hydrogen peroxide-responsive element (HRE) (between nucleotides -533 and -520). Gel mobility shift assays showed that the existence of an H(2)O(2)-inducible protein bound to the oligonucleotide of the HRE. Similar analyses showed that the heat shock activated the SOD1 promoter through the heat shock element (HSE) (between nucleotides -185 and -171). A strong specific far-shifted complex with the oligonucleotide of the HSE was observed by the treatment of heat shock. When cells were treated with PQ, a strong far-shifted complex with the HSE was observed and was competed out by the cold HSE probe, indicating that PQ also activated the SOD1 promoter through the same HSE site. It is very interesting to note that chemical and physical stresses, such as PQ and heat shock, respectively, activated the SOD1 promoter through the same cis-element HSE. These results indicate that the SOD1 was inducible by H(2)O(2) through the HRE and by PQ and heat shock through the same HSE to protect cells from oxidative hazards.

Induction of the rat Cu/Zn superoxide dismutase gene through the peroxisome proliferator-responsive element by arachidonic acid.

The Cu/Zn superoxide dismutase (SOD1) catalyzes the dismutation of superoxide radicals produced from biological oxidation and environmental stresses. From the sequence analysis of transcription factor binding sites, the peroxisome proliferator-responsive element (PPRE) was located between nt -797 and -786 of the 5'-flanking sequence of the SOD1 gene. A promoter region was fused to a chloramphenicol acetyl-transferase gene, and the resultant construct was transiently transfected into HepG2 cells. The expression of the SOD1 gene was induced by arachidonic acid (AA). Functional analyses of the PPRE site by deletion, mutations, and the heterologous promoter system confirmed the induction of the SOD1 gene by AA through the PPRE site. Gel mobility shift assays showed AA inducible binding of the peroxisome proliferator-activated receptor (PPAR) to the PPRE. The intensity of PPAR binding was also increased by the treatment of retinoic acid (RA) and 9-cis retinoic acid (9-cis RA). These results suggest that the PPRE participates in the induction of the rat SOD1 gene by the peroxisome proliferator.

Positive and negative regulatory elements in the upstream region of the rat Cu/Zn-superoxide dismutase gene.

Cu/Zn-superoxide dismutase (SOD1) catalyses the dismutation of superoxide radicals and neutralizes the oxidative effects of various chemicals. Deletion analysis of the upstream region of the rat SOD1 gene revealed that the promoter contains a positive regulatory element (PRE) and a negative regulatory element (NRE), which encompass the regions from -576 to -412 and from -412 to -305 respectively from the site of initiation of transcription. These DNA elements showed enhancer and silencer activities respectively in the natural context and in a heterologous promoter system. Using an electrophoretic-mobility-shift assay and a supershift assay with a specific antibody, the cis-elements of the PRE and NRE were identified as binding sites for transcription factors Elk1 and YY1 (Ying-Yang 1) respectively. Consistent with the presumed roles of the PRE and NRE, Elk1 increased SOD1 gene transcription about 4-5-fold, whereas YY1 exerted a negative effect of about 6-fold. Mutations of the Elk1- and YY1-binding sites led to diminution and elevation respectively of transcriptional activities, both in the natural context and in heterologous promoter systems. These results suggest that the transcription factors Elk1 and YY1, binding in the PRE and NRE respectively, co-ordinate the expression of the SOD1 gene.

Xenobiotic-responsive element for the transcriptional activation of the rat Cu/Zn superoxide dismutase gene.

Cu/Zn superoxide dismutase (SOD1) catalyzes the dismutation of superoxide radicals produced from biological oxidation and environmental stresses. A number of xenobiotics are toxic because they generate free radicals, such as superoxide and hydroxyl radicals, through a redox cycle. The xenobiotic responsive element (XRE) was located between the nt -268 and -262 region of the 5'-flanking sequence of the SOD1 gene. Functional analyses of this element by deletion, mutations, and heterologous promoter systems confirmed that the expression of the SOD1 gene was induced by a xenobiotic through the XRE. Gel mobility shift assays showed the xenobiotic inducible binding of the receptor-ligand complex to XRE. The cytoplasmic fraction from nontreated HepG2 cells also contains the factor as a cryptic form and prominently reveals its DNA-binding activity by incubation with betaNF in vitro. These results suggest that the XRE participates in the induction of the rat SOD1 gene by xenobiotics.

Overexpression and simple purification of human superoxide dismutase (SOD1) in yeast and its resistance to oxidative stress.

The structural gene of human Cu/Zn superoxide dismutase (hSOD1) was cloned into a yeast expression vector containing the promoter of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. The recombinant plasmid produced hSOD1 (20 kDa), about 6% of the total cellular protein, and the expressed hSOD1 was enzymatically active. The hSOD1 was purified from the cultured yeast by ammonium sulfate-methanol extraction and DEAE-cellulose column chromatography. This relatively simple purification method produced a single band on analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The amount of hSOD1 appeared to be considerably increased in cultures of higher cell density. The yeast overexpressing hSOD1 appeared to be more resistant to oxidative stresses such as paraquat, menadione and heat shock.

C/EBP alpha is a major activator for the transcription of rat Cu/Zn superoxide dismutase gene in liver cell.

The rat Cu/Zn superoxide dismutase (SOD1) is expressed in all tissues. Sequence analysis of the SOD1 promoter region showed that none of the cis-elements of hepatocyte-specific nuclear factors (HNF) were observed. The cis-element of C/EBP alpha in the proximal region of the SOD1 promoter and the high level of C/EBP alpha in the liver tissue led us to focus on the transcriptional regulation of the SOD1 gene by C/EBP alpha. Cotransfection assays with the plasmid expressing transcription factor C/EBP alpha showed that C/EBP alpha transactivated SOD1 gene by 27 fold. The marked transactivation and direct binding of C/EBP alpha to the SOD1 promoter were confirmed by deletion analyses and mobility shift assays. These results suggested that C/EBP alpha plays a major role in the tissue distribution of SOD1.

Transcriptional control of the Saccharomyces cerevisiae ADH1 gene by autonomously replicating sequence binding factor 1.

Autonomously replicating sequence (ARS)-binding factor 1 (ABF1) is a multifunctional protein involved in transcriptional activation and repression, as well as DNA replication, in yeast. The ADH1 gene, encoding alcohol dehydrogenase 1, contains two ABF1 consensus binding sites in the promoter and the coding regions. To examine the effect of ABF1 on expression of the ADH1 gene, we constructed an ADH1-lacZ fusion plasmid. Both ABF1 binding sites appeared to be transcriptional activators because deletions and mutations of these sites decreased transcriptional activity. The ABF1 binding sites also acted in an orientation-independent manner when a synthetic ABF1 binding site was inserted into the yeast CYC1 gene lacking its transcriptional activation region. A gel mobility shift assay showed that ABF1 bound in vitro to both ABF1 binding sites in the promoter and coding regions. In a glycerol medium the degree of activation by ABF1 was higher than in a glucose medium. The expression of ADH1 was activated synergistically by both ABF1 binding sites. These observations suggest that ABF1 transactivates the ADH1 gene through its binding sequences in both the promoter and coding regions.

Effect of growth factors on the expression of proto-oncogenes c-fos and c-myc in FRTL-5 cell line.

This study was performed to prove the hypothesis that oncogene expressions would have the same patterns with those of cellular growth to growth factors in FRTL-5 cells. Ribonucleic acids of FRTL-5 were extracted at 15', 30', 60' and 120' after administration of growth factors to quiescent FRTL-5, and blotted to the nitrocellulose membrane. They were hybridized with radiolabelled c-fos, c-myc and beta-actin probes. Hybridized dot blots were autoradiographed and the amount of radioactivity was measured by densitometry. Densitometric readings were used as the indices of oncogene expressions. Expressions of c-fos and c-myc were more prominent in combined administrations of TSH (10 mU/ml) and IGF-I (100 ng/ml) or IgG of Graves' disease (Graves' IgG; 1 mg/ml) and IGF-I than in combined administration of TSH and Graves' IgG. IgG of primary myxedema suppressed oncogene expressions by TSH or Graves' IgG, but not by IGF-I. From the above results, it was suggested that expressions of c-fos and c-myc to growth factors would have similar patterns with those of cell growth to growth factors in FRTL-5, and the actions of TSH and Graves' IgG would be manifested through same signal transduction system, but IGF-I would be manifested by its own.

The glucose-dependent transactivation activity of ABF1 on the expression of the TDH3 gene in yeast.

Autonomously replicating sequence binding factor 1 (ABF1) has been implicated in the control of a variety of gene expressions in Saccharomyces cerevisiae. In this paper evidence is presented that ABF1 is involved in the glucose-dependent expression of the TDH3 gene which encodes glyceraldehyde-3-phosphate dehydrogenase. ABF1 binds to consensus sites located between -420 and -250, and between +77 and +200, and acts as a transactivator in an orientation-independent manner on both upstream and downstream sites. TDH3-lacZ fusions having an ABF1 consensus motif showed glucose-dependent expression of TDH3, whereas in the abf1 mutant strain JCA35 glucose-dependent expression disappeared. These findings suggest that ABF1 functions as a glucose-dependent transactivator for the expression of the TDH3 gene.

Isolation and analysis of the rat genomic sequence encoding Cu/Zn superoxide dismutase.

A rat genomic DNA (SOD1) encoding Cu/Zn superoxide dismutase (SOD1) (superoxide; superoxide oxidoreductase, EC 1.15.1.1) was cloned and sequenced. The rat SOD1 gene consisted of five exons and four introns spanning about 6 kb. The transcription start point (tsp) was observed 93 bp upstream from the ATG codon by primer extension analysis. The 5'-flanking sequence of SOD1 contained two CCAAT box motifs, a TATA box and four GC-like boxes. In the 3'-flanking region of SOD1, a polyadenylation signal, consensus sequence YGTGTTYY, and a G/T cluster were observed. A rat identifier (ID) sequence, a repetitive element of the rat genome, was located at between 569 and 484 bp upstream from the tsp.

Changes in thyrotropin receptor antibody after subtotal thyroidectomy in Graves' disease: comparison with the degree of lymphocytic infiltration in the thyroid.

To evaluate changes in TSH receptor antibody after surgery in Graves' disease and its relationship with the degree of lymphocytic infiltration, serial serum levels of TSH receptor antibody were measured before and after the subtotal thyroidectomy in 50 patients with Graves' disease. In 22 (44%) out of 50 patients, thyrotropin binding inhibitor immunoglobulin (TBII) levels gradually decreased and disappeared completely within 12 months after surgery (TBII disappearing group). Twenty-eight (56%) patients showed persistent TBII activity and their levels were not changed until 12 months after surgery (TBII persistent group). The changes of thyroid stimulating antibody (TSab) levels were very similar to those of TBII in both groups. The thyroidal lymphocytic infiltration was more prominent in the TBII disappearing group. The degree of the decrease of TBII levels after surgery correlated with the grade of thyroidal lymphocytic infiltration. There was no significant difference of TSH receptor antibody (both TBII and TSab) levels between the thyroid and peripheral venous blood. These data suggest that the persistence or disappearance of TSH receptor antibody after surgery may reflect the difference between patients in whom the thyroid is the major site of TSH receptor antibody and those in whom additional sites of TSH receptor antibody synthesis exist.