Magnolol promotes thermogenesis and attenuates oxidative stress in 3T3-L1 adipocytes.

OBJECTIVE: The aim of this study was to explore the browning and antioxidative effects of magnolol in 3T3-L1 adipocytes, as recruitment of beige-like adipocytes (browning) by natural compounds is being considered as a promising strategy to fight against obesity. METHODS: Magnolol-induced browning effect was evaluated by determining the expression levels of specific marker genes and proteins using real-time polymerase chain reaction and immunoblotting, respectively. Induction of thermogenesis and suppression of oxidative stress in 3T3-L1 adipocytes were further validated by immunofluorescence. RESULTS: Magnolol significantly enhanced expression of a core set of brown fat-specific marker genes (Ucp1, Cd137, Prdm16, Cidea, and Tbx1) and proteins (UCP1, PRDM16, and PGC-1α). Increased expression of UCP1 and other brown fat-specific markers contributed to the browning of 3T3-L1 adipocytes possibly via activation of the AMPK, PPARγ, and protein kinase A (PKA) pathways. In addition, magnolol up-regulated key fatty acid oxidation and lipolytic markers (CPT1, ACSL1, SIRT1, and PLIN) and down-regulated lipogenic markers (FAS and SREBP1). Magnolol also reduced the production and release of reactive oxygen species. CONCLUSION: The current data suggest possible roles for magnolol in browning of white adipocytes, augmentation of lipolysis, and thermogenesis, as well as repression of oxidative stress and lipogenesis. Thus, magnolol may be explored as a potentially promising therapeutic agent for the prevention of obesity and other metabolic disorders.

Obstructive sleep apnea in postmenopausal women: a comparative study using drug induced sleep endoscopy / Apneia obstrutiva do sono em mulheres na pós-menopausa: estudo comparativo usando endoscopia do sono induzido por fármaco

Abstract Introduction: The key to successful treatment of OSAS is to individually tailor such treatment. Thus, it is very important to determine the severity of OSAS, its pattern, and the extent of collapse, by gender, age, and BMI. Objective: The objective of the study was to understand the characteristics of obstructive sleep apnea in postmenopausal women by comparing postmenopausal and premenopausal subjects, and men, using DISE. We hope that our work will help the medical community to consult on, diagnose, and treat OSAS more effectively. Methods: A total of 273 patients (195 males and 78 females) diagnosed with OSAS were enrolled. Female patients were divided into pre-menopausal (n = 41) and post-menopausal patients (n = 37). The group of post-menopausal female patients was matched with a group of male patients with similar age and body mass index (BMI). DISE findings were compared between pre-menopausal female patients and post-menopausal female patients, and also between post-menopausal female patients and male patients matched for age and BMI. Results: Upon PSG examination, post-menopausal patients (who had a significantly higher BMI than did pre-menopausal patients; 25.6 kg/m2 vs. 23.5 kg/m2; p = 0.019) tended to have a higher AHI and a lower lowest SaO2, but the differences did not attain statistical significance. With DISE analysis, post-menopausal female patients showed higher values in all obstruction sites, with significantly higher value in lateral diameter of retropalatal (1.49 vs. 0.90; p = 0.001) and retrolingual levels (1.14 vs. 0.61; p = 0.003) compared to pre-menopausal females patients. Post-menopausal female patients showed significantly more retrolingual collapse (antero-posterior, AP, p ≤ 0.0001, and lateral diameter, p = 0.042) in the lower BMI group (BMI < 25) and more concentric retropalatal collapse (lateral diameter, p = 0.017 and tonsillar obstruction, p = 0.003) in higher BMI group (BMI ≥ 25) than BMI and age matched male patients. Conclusion: Post-menopausal female patients showed a different pattern of airway obstruction compared to pre-menopausal female patients and male patients matched for age and BMI based on DISE findings.

Resumo Introdução: A chave para o sucesso do tratamento da síndrome da apneia obstrutiva do sono (SAOS) é adaptar individualmente esse tratamento. Assim, é muito importante determinar a gravidade da SAOS, seu padrão e a medida do colapso, por sexo, idade e IMC. Objetivo: O objetivo do estudo foi compreender as características da apneia obstrutiva do sono em mulheres na pós-menopausa, comparando estas características entre mulheres na pós-menopausa e pré-menopausa, e homens, utilizando endoscopia do sono induzido por fármacos (DISE). Esperamos que o nosso estudo ajude a comunidade médica a diagnosticar e tratar a SAOS de maneira mais eficaz. Método: Foram recrutados 273 pacientes (195 do sexo masculino e 78 do feminino) com diagnóstico de SAOS. As pacientes do sexo feminino foram divididas em pacientes na pré-menopausa (n = 41) e na pós-menopausa (n = 37). O grupo de pacientes do sexo feminino na pós-menopausa foi pareada com um grupo de pacientes do sexo masculino com idade e Índice de Massa Corporal (IMC) semelhantes. Os achados da DISE foram comparados entre as pacientes do sexo feminino na pré-menopausa e as pacientes do sexo feminino pós-menopausa e também entre pacientes do sexo feminino na pós-menopausa e pacientes do sexo masculino pareados por idade e IMC. Resultados: Ao exame de PSG, as pacientes na pós-menopausa (que tinham um IMC significativamente maior do que as pacientes na pré-menopausa; 25,6 vs. 23,5 kg/m2; p = 0,019) tenderam a ter um IAH superior e uma saturação arterial de oxigênio (SaO2) mínima menor, mas as diferenças não atingiram significância estatística. Na análise do DISE, pacientes do sexo feminino pós-menopausa apresentaram valores mais elevados em todos os locais de obstrução, com um valor significativamente maior de diâmetro lateral dos níveis retropalatal (1,49 vs. 0,90; p = 0,001) e retrolingual (1,14 vs. 0,61; p = 0,003) em comparação com pacientes do sexo feminino na pré-menopausa. As pacientes do sexo feminino na pós-menopausa apresentaram colapso significativamente mais retrolingual (anteroposterior, AP, p ≤ 0,0001 e diâmetro lateral, p = 0,042) no grupo de IMC menor (IMC < 25) e colapso retropalatal mais concêntrico (diâmetro lateral, p = 0,017 e obstrução tonsilar, p = 0,003) no grupo de maior IMC (IMC ≥ 25) do que pacientes do sexo masculino pareados por IMC e idade. Conclusão: Com base nos achados do DISE, as pacientes do sexo feminino na pós-menopausa apresentaram um padrão diferente de obstrução das vias respiratórias em comparação com pacientes do sexo feminino na pré-menopausa e com os pacientes do sexo masculino pareados por idade e IMC.

Upper airway structural changes induced by CPAP in OSAS patients: a study using drug-induced sleep endoscopy.

We studied upper airway structural changes induced by continuous positive airway pressure (CPAP) in obstructive sleep apnea syndrome (OSAS) patients using drug-induced sleep endoscopy (DISE). This prospective study was conducted at an academic secondary referral center. In total, 28 male OSAS patients (mean age 41.1 years) with only retropalatal level obstructions were enrolled. Measurements of the obstruction site were obtained in two steps: first a measurement was taken of the obstruction site in accordance with sleep apnea, then, a measurement was taken of the obstruction site in accordance with DISE-assisted CPAP titration, including quantitative changes in the occlusion site before and after CPAP in pixel format using an area calculation program. There was a tendency for persistent closing in cases of antero-posterior (AP) obstruction versus cases of lateral (Lat) obstruction in the CPAP titration. Lat obstructions showed a tendency to be wider than AP obstructions in the quantitative analysis. These results show that the pattern and degree of airway expansion after CPAP differ in accordance with the obstruction site.

Analysis of obstruction site in obstructive sleep apnea syndrome patients by drug induced sleep endoscopy.

PURPOSE: We analyzed site, pattern and degree of obstruction in Korean male obstructive sleep apnea syndrome (OSAS) patients by drug-induced sleep endoscopy (DISE). We also investigated possible links between BMI, AHI and DISE findings. MATERIALS AND METHODS: Sixty-nine male patients underwent DISE. DISE findings were reported using our classification system in which modified 'VOTE classification' - obstruction type, site of obstruction, degree of obstruction and anatomical site contributing obstruction - was reported. Associations were analyzed among the results of the polysomnography, patients' characteristics and DISE finding. RESULTS: Multilevel airway obstruction was found in 84.06% of patients and 15.94% had a unilevel obstruction. Among those with unilevel obstruction, 90.90% had retropalatal level obstruction and 9.10% had retrolingual level obstruction. Palate with lateral pharyngeal wall obstruction (49.28%) is the most common obstruction type of the retropalatal level and tongue with lateral pharyngeal wall (37.68%) is the most common obstruction type of the retrolingual level. Examining the relation between obstruction site according to body mass index (BMI) and severity of OSAS (apnea hypopnea index, AHI), the lateral pharyngeal wall had an increasing tendency associated with higher BMI and higher AHI. But the lateral pharyngeal wall of both levels was statistically significant associated with higher AHI. CONCLUSION: The majority of the Korean male OSAS patients have multilevel obstruction and according to BMI and AHI, the DISE findings indicate that the lateral pharyngeal wall is the most important anatomical site contributing to obstruction regardless of the level at which the obstruction lies.

Hypoglycemic dipeptide cyclo (His-Pro) significantly altered plasma proteome in streptozocin-induced diabetic rats and genetically-diabetic (ob/ob) mice.

The proteins in plasma perform many important functions in the body, and the protein profiles of the plasma vary under different physiological and pathological conditions. In an attempt to identify novel marker proteins for diabetes prognosis, we examined the effect of hypoglycemic dipeptide cyclo (His-Pro) (CHP) on the differential regulation of plasma proteins in streptozocin-induced diabetic rats and genetically-diabetic (ob/ob) mice. The orally-administrated CHP produced an excellent hypoglycemic effect in both animal models, lowering the average plasma glucose level by over 50 %. In the 2-DE analysis of the plasma, a total of 23 spots among 500 visualized spots were found to be differentially regulated, and they were identified by MALDI/TOF mass spectrometry. These proteins include the down-regulation of Apo E and the up-regulation of FGA, Apo A-I, Apo A-IV, and A1M in STZ-induced diabetic rats. Moreover, CHP significantly reduced the plasma protein levels of FGB, FGC, F12, C1QTNF5, and SPA3K, as well as increased the abundance of A1M, A2M, Apo E, and TTR in genetically-diabetic mice. In conclusion, alteration in the regulation of these proteins indicates that this treatment may be successful in overcoming the diabetic state. The present proteomic data can serve as the basis for the development of specific evidence-based interventions allowing for the prevention and treatment of diabetes.

Glucose tolerance and antioxidant activity of spent brewer's yeast hydrolysate with a high content of Cyclo-His-Pro (CHP).

UNLABELLED: To elevate the Cyclo-His-Pro (CHP) content in yeast, the yeast hydrolysate that was obtained from enzymatic hydrolysis was subjected to various treatments. Flavourzyme-treated hydrolysate showed the highest CHP content (674.0 µg/g) among the various proteases treatments. Ultrafiltration was selected as the best method for concentrating CHP in yeast hydrolysate, based on the yields and CHP contents. In addition, we evaluated the radical scavenge and glucose tolerance of yeast hydrolysate with a high content of CHP. Yeast hydrolysate showed intense scavenging abilities of both 1, 1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radicals. The IC(50) values of yeast hydrolysate on DPPH and ABTS radicals were 1.9 and 0.9 mg/mL, respectively. There were significant differences in glucose level between the diabetes-control and yeast hydrolysate group at 30, 60, 90, and 120 min after injection in a type 1 diabetes model (P < 0.01). Also, there were significant differences in blood glucose levels between the 2 groups at 30, 60, and 100 min after injection in the type 2 diabetes group (P < 0.05). Therefore, it is possible to use the yeast hydrolysate with high levels of CHP as an antioxidative and/or antidiabetic material for the preparation of functional foods. PRACTICAL APPLICATION: This study tried to develop a material containing a high content of CHP using yeast for possible applications of this cyclic dipeptide in the therapy of metabolic disorders. The yeast hydrolysate prepared with Flavourzyme showed a high level of CHP. The hydrolysate with a high content of CHP showed high levels of radical scavenging activities and oral glucose tolerance activity. Therefore, it is possible to use the yeast hydrolysate with high levels of CHP as an antioxidative and/or antidiabetic material for the preparation of functional foods.

Protective effect of cyclo(his-pro) on streptozotocin-induced cytotoxicity and apoptosis in vitro.

Cyclo(His-Pro) (CHP) is a naturally occurring, cyclic dipeptide structurally related to thyrotropin-releasing hormone (TRH). CHP was efficiently obtained from soybean meal by hydrolysis with flavourzyme and alcalase. In this study, the effects of CHP on streptozotocin (STZ)-induced beta-cell dysfunction and apoptosis were investigated in rat insulinoma cells (RINm5F) secreting insulin. When the RINm5F cells were treated with 2mM STZ, insulin secretion decreased to approximately 54% that of control cells. However, CHP treatment restored the insulin-secreting activity of RINm5F cells to approximately 71% that of the untreated control cells. Moreover, CHP significantly protected the cells from STZ-mediated cytotoxicity via reduction of nitric oxide (NO) production (2.3-fold) and lipid peroxidation (1.9-fold), which were induced by STZ. Moreover, CHP treatment also attenuated STZ-induced apoptotic events, such as activation of caspase-3, poly(ADP-ribose) polymerase (PARP) cleavage, and DNA fragmentation in RINm5F cells, indicating that CHP could protect the cells from apoptotic cell death induced by oxidative stress of STZ by increasing the expression of an anti-apoptotic protein, Bcl-2. These results suggest that CHP could be a candidate material for a protective and therapeutic agent against STZ-mediated cytotoxicity and apoptosis.

Functional analysis of the role of Fur in the virulence of Pseudomonas syringae pv. tabaci 11528: Fur controls expression of genes involved in quorum-sensing.

In most bacteria, Fur (ferric uptake regulator) is a crucial global regulator known to operate not only in the regulation of iron homeostasis but also in a variety of other cellular processes. In an effort to characterize the role of Fur in the virulence of plant pathogens, a fur homolog was isolated from Pseudomonas syringae pv. tabaci 11528. Phenotype assays showed that a fur deletion mutant (BL33) constitutively produced siderophore(s) and exhibited decreases in swarming motility as well as the synthesis of tabtoxin and N-acyl homoserine lactones. Consistent with the results of TLC, quantitative real-time RT-PCR of the QS associated genes psyR and psyI demonstrated that Fur up-regulates these genes at the transcriptional level. Finally, the effects of a fur mutation on plant virulence indicated that Fur-regulated traits are relevant to plant-pathogen interactions.

Cloning and expression of the cathepsin F-like cysteine protease gene in Escherichia coli and its characterization.

In this study, we have cloned a novel cDNA encoding for a papain-family cysteine protease from the Uni-ZAP XR cDNA library of the polychaete, Periserrula leucophryna. This gene was expressed in Escherichia coli using the T7 promoter system, and the protease was characterized after partial purification. First, the partial DNA fragment (498 bp) was amplified from the total RNA via RT-PCR using degenerated primers derived from the conserved region of cysteine protease. The full-length cDNA of cysteine protease (PLCP) was prepared via the screening of the Uni-ZAP XR cDNA library using the 32P-labeled partial DNA fragment. As a result, the PLCP gene was determined to consist of a 2591 bp nucleotide sequence (CDS: 173-1024 bp) which encodes for a 283-amino acid polypeptide, which is itself composed of an 59-residue signal sequence, a 6-residue propeptide, a 218-residue mature protein, and a long 3'-noncoding region encompassing 1564 bp. The predicted molecular weights of the preproprotein and the mature protein were calculated as 31.8 kDa and 25 kDa, respectively. The results of sequence analysis and alignment revealed a significant degree of sequence similarity with other eukaryotic cysteine proteases, including the conserved catalytic triad of the Cys90, His226, and Asn250 residues which characterize the C1 family of papain-like cysteine protease. The nucleotide and amino acid sequences of the novel gene were deposited into the GenBank database under the accession numbers, AY390282 and AAR27011, respectively. The results of Northern blot analysis revealed the 2.5 kb size of the transcript and ubiquitous expression throughout the entirety of the body, head, gut, and skin, which suggested that the PLCP may be grouped within the cathepsin F-like proteases. The region encoding for the mature form of the protease was then subcloned into the pT7-7 expression vector following PCR amplification using the designed primers, including the initiation and termination codons. The recombinant cysteine proteases were generated in a range of 6.3% to 12.5% of the total cell proteins in the E. coli BL21(DE3) strain for 8 transformants. The results of SDS-PAGE and Western blot analysis indicated that a cysteine protease of approximately 25 kDa (mature form) was generated. The optimal pH and temperature of the enzyme were determined to be approximately 9.5 and 35 degrees, respectively, thereby indicating that the cysteine protease is a member of the alkaline protease group. The evaluation of substrate specificity indicated that the purified protease was more active towards Arg-X or Lys-X and did not efficiently cleave the substrates with non-polar amino acids at the P1 site. The PLCP evidenced fibrinolytic activity on the plasminogen-free fibrin plate test.

Characterization, cloning and expression of the ferritin gene from the Korean polychaete, Periserrula leucophryna.

Ferritin is a major eukaryotic protein and in humans is the protein of iron storage. A partial gene fragment of ferritin (255 bp) taken from the total RNA of Periserrula leucophryna, was amplified by RT-PCR using oligonucleotide primers designed from the conserved metal binding domain of eukaryotic ferritin and confirmed by DNA sequencing. Using the 32P-labeled partial ferritin cDNA fragment, 28 different clones were obtained by the screening of the P. leucophryna cDNA library prepared in the Uni-ZAP XR vector, sequenced and characterized. The longest clone was named the PLF (Periserrula leucophryna ferritin) gene and the nucleotide and amino acid sequences of this novel gene were deposited in the GenBank databases with accession numbers DQ207752 and ABA55730, respectively. The entire cDNA of PLF clone was 1109 bp (CDS: 129-653), including a coding nucleotide sequence of 525 bp, a 5'-untranslated region of 128 bp, and a 3'-noncoding region of 456 bp. The 5'-UTR contains a putative iron responsive element (IRE) sequence. Ferritin has an open reading frame encoding a polypeptide of 174 amino acids including a hydrophobic signal peptide of 17 amino acids. The predicted molecular weights of the immature and mature ferritin were calculated to be 20.3 kDa and 18.2 kDa, respectively. The region encoding the mature ferritin was subcloned into the pT7-7 expression vector after PCR amplification using the designed primers and included the initiation and termination codons; the recombinant clones were expressed in E. coli BL21(DE3) or E. coli BL21(DE3)pLysE. SDS-PAGE and western blot analysis showed that a ferritin of approximately 18 kDa (mature form) was produced and that by iron staining in native PAGE, it is likely that the recombinant ferritin is correctly folded and assembled into a homopolymer composed of a single subunit.

Overexpression of human erythropoietin (EPO) affects plant morphologies: retarded vegetative growth in tobacco and male sterility in tobacco and Arabidopsis.

Erythropoietin (EPO) is a glycoprotein used for curing human anemia by regulating the differentiation of erythroid progenitors and the production of red blood cells. To examine the expression of recombinant EPO in plants, pPEV-EP21, in which human epo cDNA under the control of the CaMV 35S promoter, was introduced into tobacco and Arabidopsis via Agrobacterium tumefaciens-mediated transformation. The RNA expression level of epo in the transgenic lines was initially estimated by Northern blot analysis. Two transgenic lines, which exhibited a high expression level of epo mRNA determined by Northern analysis, were chosen for Western blot analysis to examine the production of EPO proteins. Those two lines, EP21-12 and EP21-14, revealed detectable bands on the immunoblot. Interestingly, constitutive expression of the human epo gene affected the morphologies in transgenic plants such that vegetative growth of transgenic tobacco was retarded, and male sterility was induced in transgenic tobacco and Arabidopsis.

Expression of the bovine growth hormone alters the root morphology in transgenic tobacco plants.

The bovine growth hormone (bGH) is a natural peptide hormone that controls the differentiation, growth and metabolism, and is produced in the pituitary gland of cows. For the production of bGH from plants, two different bgh clones, of which the pGAbGH1 contaions only mature peptide sequences and the pGAbGH15 contains signal sequences and the first intron, as well as mature peptide sequences, were used. Those bghs under the control of the CaMV 35S promoter and NOS terminator were introduced to tobacco plants via Agrobacterium tumefaciens-mediated transformation. By PCR analyses using bgh and nptII specific primers, 17 and 21 putative transformants were respectively selected from pGAbGH1- and pGAbGH15-transformed tobacco plants. Northern blot analysis showed that the most of the transgenic lines expressed the bgh mRNA. Western blot analysis revealed that the pGAbGH1-transformed tobaccos produced recombinant bGH, but pGAbGH15-transformed ones did not produce the protein. Interestingly, some morphological changes were observed in the roots of transgenic tobacco plants. The transgenic tobacco plants had thick and short roots containing few root hairs in contrast to the non-transformed wild type plants.