Dihydroxylation of dehydroepiandrosterone in positions 7α and 15α by mycelial fungi

The ability of 485 fungal strains is studied for catalysis of the process of 7α, 15α-dihydroxylation of dehydroepiandrosterone (DHEA, 3β-hydroxy-5-androstene-17-one), a key intermediate of the synthesis of physiologically active compounds. The ability for the formation of 3β, 7α, 15α-trihydroxy-5-androstene-17-one (7α, 15α-diOH-DHEA) was found for the first time for representatives of 12 genera, eight families, and six orders of ascomycetes, eight genera, four families, and one order of zygomycetes, one genus, one family, and one order of basidiomycetes, and four genera of mitosporic fungi. The most active strains are found among genera Acremonium, Gibberella, Fusarium, and Nigrospora. In the process of transformation of DHEA (2 g/l) by strains of Fusarium oxysporum VKM F-1600 and Gibberella zeae BKM F-2600, the molar yield was 63 and 68%, respectively. Application of the revealed active strains of microorganisms opens prospects for the efficient production of key intermediates of synthesis of modern medical preparations.     

Role of PPARα and HNF4α in Stress-Mediated Alterations in Lipid Homeostasis

Stress is a risk factor for several cardiovascular pathologies. PPARα holds a fundamental role in control of lipid homeostasis by directly regulating genes involved in fatty acid transport and oxidation. Importantly, PPARα agonists are effective in raising HDL-cholesterol and lowering triglycerides, properties that reduce the risk for cardiovascular diseases. This study investigated the role of stress and adrenergic receptor (AR)-related pathways in PPARα and HNF4α regulation and signaling in mice following repeated restraint stress or treatment with AR-antagonists administered prior to stress to block AR-linked pathways. Repeated restraint stress up-regulated Pparα and its target genes in the liver, including Acox, Acot1, Acot4, Cyp4a10, Cyp4a14 and Lipin2, an effect that was highly correlated with Hnf4α. In vitro studies using primary hepatocyte cultures treated with epinephrine or AR-agonists confirmed that hepatic AR/cAMP/PKA/CREB- and JNK-linked pathways are involved in PPARα and HNF4α regulation. Notably, restraint stress, independent of PPARα, suppressed plasma triglyceride levels. This stress-induced effect could be attributed in part to hormone sensitive lipase activation in the white adipose tissue, which was not prevented by the increased levels of perilipin. Overall, this study identifies a mechanistic basis for the modification of lipid homeostasis following stress and potentially indicates novel roles for PPARα and HNF4α in stress-induced lipid metabolism.     

HIF-1α protein is upregulated in HIF-2α depleted cells via enhanced translation

The hypoxia-inducible factors HIF-1 and HIF-2 are primarily regulated via stabilization of their respective α-subunits under hypoxic conditions. Previously, compensatory upregulation of one HIF-α-subunit upon depletion of the other α-subunit was described, yet the underlying mechanism remained elusive. Here we provide evidence that enhanced HIF-1α protein expression in HIF-2α knockdown (k/d) cells neither results from elevated HIF-1α mRNA expression, nor from increased HIF-1α protein stability. Instead, we identify enhanced HIF-1α translation as molecular mechanism. Moreover, we found elevated levels of the RNA-binding protein HuR and provide evidence that HuR is critical for the compensatory HIF-1α regulation in HIF-2α k/d cells.     

Skeletal muscle PGC-1α is required for maintaining an acute LPS-induced TNFα response

Many lifestyle-related diseases are associated with low-grade inflammation and peroxisome proliferator activated receptor γ coactivator (PGC)-1α has been suggested to be protective against low-grade inflammation. However, whether these anti-inflammatory properties affect acute inflammation is not known. The aim of the present study was therefore to investigate the role of muscle PGC-1α in acute inflammation. Quadriceps muscles were removed from 10-week old whole body PGC-1α knockout (KO), muscle specific PGC-1α KO (MKO) and muscle-specific PGC-1α overexpression mice (TG), 2 hours after an intraperitoneal injection of either 0.8 μg LPS/g body weight or saline. Basal TNFα mRNA content was lower in skeletal muscle of whole body PGC-1α KO mice and in accordance TG mice showed increased TNFα mRNA and protein level relative to WT, indicating a possible PGC-1α mediated regulation of TNFα. Basal p65 phosphorylation was increased in TG mice possibly explaining the elevated TNFα expression in these mice. Systemically, TG mice had reduced basal plasma TNFα levels compared with WT suggesting a protective effect against systemic low-grade inflammation in these animals. While TG mice reached similar TNFα levels as WT and showed more marked induction in plasma TNFα than WT after LPS injection, MKO PGC-1α mice had a reduced plasma TNFα and skeletal muscle TNFα mRNA response to LPS. In conclusion, the present findings suggest that PGC-1α enhances basal TNFα expression in skeletal muscle and indicate that PGC-1α does not exert anti-inflammatory effects during acute inflammation. Lack of skeletal muscle PGC-1α seems however to impair the acute TNFα response, which may reflect a phenotype more susceptible to infections as also observed in type 2 diabetes patients.     

Specific in vitro phosphorylation of plant eIF2α by eukaryotic eIF2α kinases

Phosphorylation of the subunit of eukaryotic initiation factor 2 (eIF2) is known to be an important translational control mechanism in all eukaryotes with the major exception of plants. Regulation of mammalian and yeast eIF2 activity is directly governed by specific phosphorylation on Ser-51. We now demonstrate that recombinant wheat wild-type (51S) but not mutant 51-Ala (51A) protein is phosphorylated by human PKR and yeast GCN2, which are defined eIF2 kinases. Further, only wheat wild-type eIF2 is a substrate for plant-encoded, double-stranded RNA-dependent kinase (pPKR) activity. Plant PKR and GCN2 phosphorylate recombinant yeast eIF2 51S but not the 51A mutant demonstrating that pPKR has recognition site capability similar to established eIF2 kinases. A truncated version of wild-type wheat eIF2 containing 51S but not the KGYID motif is not phosphorylated by either hPKR or pPKR suggesting that this putative eIF2 kinase docking domain is essential for phosphorylation. Taken together, these results demonstrate the homology among eukaryotic eIF2 species and eIF2 kinases and support the presence of a plant eIF2 phosphorylation pathway.     

TNF-α 在肿瘤中的作用

肿瘤坏死因子是一个特别的,并具有多重功能的细胞因子,它在免疫调节,炎症反应,机体防御当中起着关键的作用。根据不同的细胞微环境,肿瘤坏死因子可以诱导多种反应,例如凋亡,坏死,血管生成,免疫细胞激活,细胞分化,细胞迁移。TNF在肿瘤当中是一把双刃剑。一方面,TNF是一个内源性的肿瘤促进因素,因为TNF可以刺激肿瘤细胞生长,增殖,侵袭,转移,血管生成。另一方面,TNF具有杀肿瘤细胞的作用。因此,如果可以调控肿瘤坏死因子的功能,将为癌症的治疗提供可能。     

Differential roles of Sirt1 in HIF-1α and HIF-2α mediated hypoxic responses

Hypoxia-inducible factors 1α and 2α (HIF-1α and HIF-2α) determine cancer cell fate under hypoxia. Despite the similarities of their structures, HIF-1α and HIF-2α have distinct roles in cancer growth under hypoxia, that is, HIF-1α induces growth arrest whereas HIF-2α promotes cell growth. Recently, sirtuin 1 (Sirt1) was reported to fine-tune cellular responses to hypoxia by deacetylating HIF-1α and HIF-2α. Yet, the roles of Sirt1 in HIF-1α and HIF-2α functions have been controversial. We here investigated the precise roles of Sirt1 in HIF-1α and HIF-2α regulations. Immunological analyses revealed that HIF-1α K674 and HIF-2α K741 are acetylated by PCAF and CBP, respectively, but are deacetylated commonly by Sirt1. In the Gal4 reporter systems, Sirt1 was found to repress HIF-1α activity constantly in ten cancer cell-lines but to regulate HIF-2α activity cell type-dependently. Moreover, Sirt1 determined cell growth under hypoxia depending on HIF-1α and HIF-2α. Under hypoxia, Sirt1 promoted cell proliferation of HepG2, in which Sirt1 differentially regulates HIF-1α and HIF-2α. In contrast, such an effect of Sirt1 was not shown in HCT116, in which Sirt1 inactivates both HIF-1α and HIF-2α because conflicting actions of HIF-1α and HIF-2α on cell growth may be offset. Our results provide a better understanding of the roles of Sirt1 in HIF-mediated hypoxic responses and also a basic concept for developing anticancer strategy targeting Sirt1.     

BDNF and TNF-α polymorphisms in memory

Here, we investigate the genetic basis of human memory in healthy individuals and the potential role of two polymorphisms, previously implicated in memory function. We have explored aspects of retrospective and prospective memory including semantic, short term, working and long-term memory in conjunction with brain derived neurotrophic factor (BDNF) and tumor necrosis factor-alpha (TNF-α). The memory scores for healthy individuals in the population were obtained for each memory type and the population was genotyped via restriction fragment length polymorphism for the BDNF rs6265 (Val66Met) SNP and via pyrosequencing for the TNF-α rs113325588 SNP. Using univariate ANOVA, a significant association of the BDNF polymorphism with visual and spatial memory retention and a significant association of the TNF-α polymorphism was observed with spatial memory retention. In addition, a significant interactive effect between BDNF and TNF-α polymorphisms was observed in spatial memory retention. In practice visual memory involves spatial information and the two memory systems work together, however our data demonstrate that individuals with the Val/Val BDNF genotype have poorer visual memory but higher spatial memory retention, indicating a level of interaction between TNF-α and BDNF in spatial memory retention. This is the first study to use genetic analysis to determine the interaction between BDNF and TNF-α in relation to memory in normal adults and provides important information regarding the effect of genetic determinants and gene interactions on human memory.     

Lysine-deficient lymphotoxin-α mutant for site-specific PEGylation

The cytokine lymphotoxin-α (LTα) is a promising anticancer agent; however, its instability currently limits its therapeutic potential. Modification of proteins with polyethylene glycol (PEGylation) can improve their in vivo stability, but PEGylation occurs randomly at lysine residues and the N-terminus. Therefore, PEGylated proteins are generally heterogeneous and have lower bioactivity than their non-PEGylated counterparts. Previously, we created phage libraries expressing mutant LTαs in which the lysine residues of wild-type LTα (wtLTα) were substituted for other amino acids. Here, we attempted to create a lysine-deficient mutant LTα with about the same bioactivity as wtLTα by using these libraries and site-specific PEGylation of the N-terminus. We isolated a lysine-deficient mutant LTα, LT-K0, with almost identical bioactivity to that of wtLTα against mouse LM cells. The bioactivity of wtLTα decreased to 10% following random PEGylation, whereas that of LT-K0 decreased to 50% following site-specific PEGylation; PEGylated LT-K0 retained five times the bioactivity of randomly PEGylated wtLTα. These results suggest that site-specific PEGylated LT-K0 may be useful in cancer therapy.     

Structures of the PKA RIα Holoenzyme with the FLHCC Driver J-PKAcα or Wild-Type PKAcα

1. Download high-res image (253KB)
2. Download full-size image

     

Antisense Knockdown of PKC-α Using LNA-Oligos

Abstract

Full-length and 4 nucleotides truncated Locked Nucleic Acid (LNA) modifications of ISIS 3521 were compared for antisense properties in a cellular assay. ISIS 3521 is a 20-mer phosphorothioate designed to hybridise to human protein kinase C-α (PKC-α) mRNA and is currently submitted to clinical trials against cancer. We report that LNA can potentate this antisense oligo and retain the antisense potential with shorter oligos.     

PPAR-α Expression Inversely Correlates with Inflammatory Cytokines IL-1β and TNF-α in Aging Rats

Dehydroepiandrosterone (DHEAS) was given the name “fountain of youth” in reference to its beneficial properties in memory, cognition and aging. Cultured cell studies showed that DHEAS may mediate its action by counteracting aging-associated inflammation via PPAR-α activation. In the present study, we demonstrated an age-dependent increase in IL-1β and TNF-α expression in the brain and the spleen of aging rats, while PPAR-α expression was decreased in the spleen of 18 month-old rats. Oral treatment with DHEAS increased PPAR-α mRNA in 3 month-old rats and decreased PPAR-α protein expression in 18 month-old rats in the spleen. In contrast, DHEAS did not alter cytokine expression in spleen and brain of the three age groups. These findings underline a differential role for DHEAS in PPAR-α expression that is age-dependent, and also, that beneficial effects of DHEAS on cognitive function are unlikely mediated by a decrease in cytokine expression.     

Anti-TNFα domain antibody construct CEP-37247

We report preclinical data for CEP-37247, the first human framework domain antibody construct to enter the clinic. At approximately 11 – 13kDa, domain antibodies or dAbs are the smallest antibody domain able to demonstrate the antigen-recognition function of an antibody, e.g. high selectivity and affinity for target antigen. CEP-37247 is a bivalent anti-tumor necrosis factor (TNF)α domain antibody protein construct combining the antigen-recognition function of a dAb with the pharmacological advantages of an antibody Fc region. As a homodimer, with each chain comprising V_L dAb, truncated C_H1, hinge, C_H2 and C_H3 domains, CEP-37247 has a molecular mass of approximately 78kDa, which is about half the size of a conventional IgG molecule. Surface plasmon resonance data demonstrate that CEP-37247 possesses high selectivity and affinity for TNFα. CEP-37247 is a potent neutralizer of TNFα activity in vitro in the L929 TNF-mediated cytotoxicity assay. In a human TNFα-over-expressing mouse model of polyarthritis, CEP-37247 prevents development of disease, and is at least as effective as the marketed product etanercept. Fc functionality is intact – CEP-37247 is capable of mediating antibody-dependent cell-mediated cytotoxicity and has a circulating half-life of approximately 4.5 days in cynomolgus macaques. Given the favorable properties outlined above, and its high expression levels (approaching 7 g/L) in a CHOK1 based-expression system, CEP-37247 is progressing into the clinic, where other potential advantages such as enhanced efficacy due to improved tissue distribution, and beneficial immunogenicity profile, will be evaluated.     

Interferon-α (IFNα) neurotoxicity

Clinical studies indicate that increased central nervous system (CNS) interferon-alpha (IFNα) is associated with cognitive dysfunction in a wide variety of conditions. This has perhaps been best studied in HIV-associated neurocognitive disorders (HAND). These findings on IFNα neurotoxicity have been corroborated in animal studies. Probably the best demonstration of the neurotoxicity of IFNα was through the use of a mouse model of HAND, where it was shown that blocking IFNα with neutralizing antibodies prevented behavioral deficits and associated histopathological effects. In vitro studies have demonstrated a dose dependent, detrimental effect of IFNα on neuronal dendrites. Development of therapeutics that block IFNα may prove to be an effective treatment of HAND and other inflammatory conditions where there is increased CNS IFNα.