Catecholamines, growth hormone, cortisol, insulin, and sex hormones in anaerobic and aerobic exercise

Seventeen male physical education students performed three types of treadmill exercise: (1) progressive exercise to exhaustion, (2) prolonged exercise of 50 min duration at the anaerobic threshold of 4 mmol . l-1 blood lactate (AE), (3) a single bout of short-term high-intensity exercise at 156% of maximal exercise capacity in the progressive test, leading to exhaustion within 1.5 min (ANE). Immediately before and after ANE and before, during, and after AE adrenaline, noradrenaline, growth hormone, cortisol, insulin, testosterone, and oestradiol were determined in venous blood, and glucose and lactate were determined in arterialized blood from the earlobe. Adrenaline and noradrenaline increased 15 fold during ANE and 3--4 fold and 6--9 fold respectively during AE. The adrenaline/noradrenaline ratio was 1 : 3 during ANE and 1 : 10 during AE. Cortisol increased by 35% in ANE (12% of which appeared in the postexercise period) and 54% in AE. Insulin increased during ANE but decreased during AE. Testosterone and oestradiol increased by 14% and 16% during ANE and by 22% and 28% during AE. The results point to a markedly higher emotional stress and higher sympatho-adrenal activity in anaerobic exercise. Growth hormone and cortisol appear to be the more affected by intense prolonged exercise. Taking plasma volume changes and changes of metabolic clearance rates into consideration, neither of the exercise tests appeared to affect secretion of testosterone and oestradiol.     

RNA binding protein 24 regulates the translation and replication of hepatitis C virus

The secondary structures of hepatitis C virus (HCV) RNA and the cellular proteins that bind to them are important for modulating both translation and RNA replication. However, the sets of RNA-binding proteins involved in the regulation of HCV translation, replication and encapsidation remain unknown. Here, we identified RNA binding motif protein 24 (RBM24) as a host factor participated in HCV translation and replication. Knockdown of RBM24 reduced HCV propagation in Huh7.5.1 cells. An enhanced translation and delayed RNA synthesis during the early phase of infection was observed in RBM24 silencing cells. However, both overexpression of RBM24 and recombinant human RBM24 protein suppressed HCV IRES-mediated translation. Further analysis revealed that the assembly of the 80S ribosome on the HCV IRES was interrupted by RBM24 protein through binding to the 5′-UTR. RBM24 could also interact with HCV Core and enhance the interaction of Core and 5′-UTR, which suppresses the expression of HCV. Moreover, RBM24 enhanced the interaction between the 5′- and 3′-UTRs in the HCV genome, which probably explained its requirement in HCV genome replication. Therefore, RBM24 is a novel host factor involved in HCV replication and may function at the switch from translation to replication.     

RNA-binding Motif Protein 4 Translocates to Cytoplasmic Granules and Suppresses Translation via Argonaute2 during Muscle Cell Differentiation

The RNA-binding motif protein 4 (RBM4) plays multiple roles in mRNA metabolism, including translation control. RBM4 suppresses cap-dependent translation but activates internal ribosome entry site-mediated translation. Because of its high expression level in muscle and heart, we investigated the function of RBM4 in myoblast cells. Here, we demonstrate that RBM4 is phosphorylated and translocates to the cytoplasm in mouse C2C12 cells upon cell differentiation. Notably, RBM4 is transiently deposited into cytoplasmic granules containing microtubule assembly factors as well as poly(A)⁺ RNAs. Moreover, RBM4 colocalizes with the components of micro-ribonucleoproteins, including the Argonaute2 (Ago2) protein, during muscle cell differentiation. RBM4 interacts directly with Ago2 and may recruit Ago2 to suppress translation of target mRNAs. Furthermore, RBM4 selectively associates with muscle cell-specific microRNAs and potentiates their translation repression activity by promoting micro-ribonucleoprotein association with target mRNAs. Altogether, our results suggest that RBM4 translocates to the cytoplasm and participates in translation suppression during muscle cell differentiation.     

Effect of Ascophyllum nodosum extract on growth performance, digestibility, carcass characteristics and selected intestinal microflora populations of grower–finisher pigs

The effects of an extract of the brown seaweed Ascophyllum nodosum (AN) in grower–finisher pigs are reported. In Experiment 1, the effect of dietary supplementation with increasing levels of AN extract (ANE) on growth performance, carcass characteristics and gastrointestinal microflora was investigated. A total of 360 pigs were randomly allocated, based on initial live-weight and sex, to one of four experimental treatments as follows; control diet (no ANE), control diet plus 3 g ANE/kg, control diet plus 6 g ANE/kg and control diet plus 9 g ANE/kg. These diets were fed ad libitum up to slaughter. In Experiment 2, eight male pigs were allocated to a control diet (no ANE) or the control diet plus 2.5 g ANE/kg to determine effects of ANE on coefficient of total tract apparent digestibility (CTTAD) of nutrients and nitrogen (N) balance. Supplementation with increasing levels of ANE in Experiment 1 resulted in reduced daily gain, carcass weight and kill-out yield during the combined grower–finisher period (P<0.05); however, there were no effects of treatment on feed intake, feed conversion ratio or carcass characteristics. Increasing levels of dietary ANE resulted in decreased ileal coliform counts (P<0.05). Increasing dietary ANE also tended to increase adherent lactobacilli in the colon (P=0.080) but caecal bifidobacteria declined (P<0.05). There were trends towards a linear reduction in colonic bifidobacteria (P=0.077) and towards a quadratic effect on rectal lactobacilli (P=0.077). Intestinal pH was unaffected by ANE supplementation (P>0.05). In Experiment 2, the CTTAD was unaffected by the inclusion of ANE (P>0.05). Overall, the intestinal coliform reductions obtained suggest that ANE may provide a dietary means to improve gut health and potentially reduce pathogen carriage in finishing pigs. However, the negative effects on growth performance observed in healthy animals will most likely limit the commercial use of dietary ANE as a feed additive.     

RBM19 is essential for preimplantation development in the mouse

Background  

RNA-binding motif protein 19 (RBM19, NCBI Accession # NP_083038) is a conserved nucleolar protein containing 6 conserved RNA recognition motifs. Its biochemical function is to process rRNA for ribosome biogenesis, and it has been shown to play a role in digestive organ development in zebrafish. Here we analyzed the role of RBM19 during mouse embryonic development by generating mice containing a mutation in the Rbm19 locus via gene-trap insertion.     

Acrofacial Dysostosis,Cincinnati Type,a Mandibulofacial Dysostosis Syndrome with Limb Anomalies,Is Caused by POLR1A Dysfunction

We report three individuals with a cranioskeletal malformation syndrome that we define as acrofacial dysostosis, Cincinnati type. Each individual has a heterozygous mutation in POLR1A, which encodes a core component of RNA polymerase 1. All three individuals exhibit varying degrees of mandibulofacial dysostosis, and two additionally have limb anomalies. Consistent with this observation, we discovered that polr1a mutant zebrafish exhibited cranioskeletal anomalies mimicking the human phenotype. polr1a loss of function led to perturbed ribosome biogenesis and p53-dependent cell death, resulting in a deficiency of neural-crest-derived skeletal precursor cells and consequently craniofacial anomalies. Our findings expand the genotypic and phenotypic heterogeneity of congenital acrofacial disorders caused by disruption of ribosome biogenesis.     

RBM10: Harmful or helpful‐many factors to consider

RBM10 is an RNA binding motif (RBM) protein expressed in most, if not all, human and animal cells. Interest in RBM10 is rapidly increasing and its clinical importance is highlighted by its identification as the causative agent of TARP syndrome, a developmental condition that significantly impacts affected children. RBM10's cellular functions are beginning to be explored, with initial studies demonstrating a tumor suppressor role. Very recently, however, contradictory results have emerged, suggesting a tumor promoter role for RBM10. In this review, we describe the current state of knowledge on RBM10, and address this dichotomy in RBM10 function. Furthermore, we discuss what may be regulating RBM10 function, particularly the importance of RBM10 alternative splicing, and the relationship between RBM10 and its paralogue, RBM5. As RBM10‐related work is gaining momentum, it is critical that the various aspects of RBM10 molecular biology revealed by recent studies be considered moving forward. It is only if these recent advances in RBM10 structure and function are considered that a clearer insight into RBM10 function, and the disease states with which RBM10 mutation is associated, will be gained.     

Human RBM28 protein is a specific nucleolar component of the spliceosomal snRNPs

The biogenesis of spliceosomal small nuclear RNAs (snRNAs) involves organized translocations between the cytoplasm and certain nuclear domains, such as Cajal bodies and nucleoli. Here we identify human RBM28 protein as a novel snRNP component, based on affinity selection of U6 small nuclear ribonucleoprotein (snRNP). As shown by immunofluorescence, RBM28 is a nucleolar protein. Anti-RBM28 immunoprecipitation from HeLa cell lysates revealed that this protein specifically associates with U1, U2, U4, U5, and U6 snRNAs. Our data provide the first evidence that RBM28 is a common nucleolar component of the spliceosomal ribonucleoprotein complexes, possibly coordinating their transition through the nucleolus.     

Infection-Triggered Familial or Recurrent Cases of Acute Necrotizing Encephalopathy Caused by Mutations in a Component of the Nuclear Pore, RANBP2

Acute necrotizing encephalopathy (ANE) is a rapidly progressive encephalopathy that can occur in otherwise healthy children after common viral infections such as influenza and parainfluenza. Most ANE is sporadic and nonrecurrent (isolated ANE). However, we identified a 7 Mb interval containing a susceptibility locus (ANE1) in a family segregating recurrent ANE as an incompletely penetrant, autosomal-dominant trait. We now report that all affected individuals and obligate carriers in this family are heterozygous for a missense mutation (c.1880C→T, p.Thr585Met) in the gene encoding the nuclear pore protein Ran Binding Protein 2 (RANBP2). To determine whether this mutation is the susceptibility allele, we screened controls and other patients with ANE who are unrelated to the index family. Patients from 9 of 15 additional kindreds with familial or recurrent ANE had the identical mutation. It arose de novo in two families and independently in several other families. Two other patients with familial ANE had different RANBP2 missense mutations that altered conserved residues. None of the three RANBP2 missense mutations were found in 19 patients with isolated ANE or in unaffected controls. We conclude that missense mutations in RANBP2 are susceptibility alleles for familial and recurrent cases of ANE.     

Structural basis of the phosphorylation dependent complex formation of neurodegenerative disease protein Ataxin-1 and RBM17

Spinocerebellar Ataxia Type1 (SCA1) is a dominantly inherited neurodegenerative disease and belongs to polyglutamine expansion disorders. The polyglutamine expansion in Ataxin-1 (ATXN1) is responsible for SCA1 pathology. ATXN1 forms at least two distinct complexes with Capicua (CIC) or RNA-binding motif protein 17 (RBM17). The wild-type ATXN1 dominantly forms a complex with CIC and the polyglutamine expanded form of ATXN1 favors to form a complex with RBM17. The phosphorylation of Ser776 in ATXN1 is critical for SCA1 pathology and serves as a binding platform for RBM17. However, the molecular basis of the phospho-specific binging of ATXN1 to RBM17 is not delineated. Here, we present the modeled structure of RBM17 bound to the phosphorylated ATXN1 peptide. The structure reveals the phosphorylation specific interaction between ATXN1 and RBM17 through a salt-bridge network. Furthermore, the modeled structure and the interactions between RBM17 and ATXN1 were validated through mutagenesis study followed by Surface Plasmon Resonance binding experiments. This work delineates the molecular basis of the interaction between RBM17 and the phosphorylated form of ATXN1, which is critical for SCA1 pathology. Furthermore, the structure of RBM17 and pATXN1 peptide might be utilized to target RBM17–ATXN1 interaction to modulate SCA1 pathogenesis.     

RBM4 interacts with an intronic element and stimulates tau exon 10 inclusion

Tau protein, which binds to and stabilizes microtubules, is critical for neuronal survival and function. In the human brain, tau pre-mRNA splicing is regulated to maintain a delicate balance of exon 10-containing and exon 10-skipping isoforms. Splicing mutations affecting tau exon 10 alternative splicing lead to tauopathies, a group of neurodegenerative disorders including dementia. Molecular mechanisms regulating tau alternative splicing remain to be elucidated. In this study, we have developed an expression cloning strategy to identify splicing factors that stimulate tau exon 10 inclusion. Using this expression cloning approach, we have identified a previously unknown tau exon 10 splicing regulator, RBM4 (RNA binding motif protein 4). In cells transfected with a tau minigene, RBM4 overexpression leads to an increased inclusion of exon 10, whereas RBM4 down-regulation decreases exon 10 inclusion. The activity of RBM4 in stimulating tau exon 10 inclusion is abolished by mutations in its RNA-binding domain. A putative intronic splicing enhancer located in intron 10 of the tau gene is required for the splicing stimulatory activity of RBM4. Immunohistological analyses reveal that RBM4 is expressed in the human brain regions affected in tauopathy, including the hippocampus and frontal cortex. Our study demonstrates that RBM4 is involved in tau exon 10 alternative splicing. Our work also suggests that down-regulating tau exon 10 splicing activators, such as RBM4, may be of therapeutic potential in tauopathies involving excessive tau exon 10 inclusion.     

Oxidative damage to DNA induced by areca nut extract

In Taiwan, people chew bete; quid which contains tender areca nut with husk. In other countries, people prefer ripe and dried areca nut without husk. In this study, we compared the reactive oxygen species-induced oxidative DNA damage in isolated DNA and CHO-K1 cells between treatments with tender areca nut extract (ANE) and ripe ANE. Incubation of these two ANE preparations with isolated DNA generated 8-hydroxy-2′-deoxyguanosine (8-OH-dG) in an alkaline environment in a dose-dependent manner. Ripe ANE generated higher levels of 8-OH-dG compared to tender ANE. The addition of iron(II) (100 μM) resulted in 1.4- and 3.1-fold increases of 8-OH-dG when incubated with 1 mg/ml each of tender and ripe ANE. In testing the effect of ANE to cellular DNA, CHO-K1 cells were used for its documented sensitivity to reactive oxygen species. In CHO-K1 cells, ripe ANE was more cytotoxic than tender ANE following an 18-h incubation. The cytotoxicity to CHO-K1 cells was positively correlated with the formation of 8-OH-dG following tender (r = 0.97) and ripe (r = 0.91) ANE treatment. Addition of the iron chelating agent o-phenanthroline (10 and 20 μM) to cells prior to ripe ANE exposure significantly increased (p < 0.05) the survival of CHO-K1 cells. In addition, ripe ANE induced dichlorofluorescein-mediated fluorescence which indicated the formation of hydrogen peroxide in CHO-K1 cells. In conclusion, this study demonstrated that ANE-induced oxidative damage to isolated and cellular DNA which may result from the generation of hydrogen peroxide, and iron may serve as a catalyst in this process. Furthermore, ripe ANE generated higher oxidative DNA damage levels compared to tender ANE.     

Genetic damage in cultured human keratinocytes stressed by long-term exposure to areca nut extracts

Chewing betel quid (BQ) is a popular habit worldwide. A causal association between BQ chewing and oral cancer has been well documented. Emerging evidence indicates that sustained exposure to stress induces epigenetic reprogramming of some mammalian cells and increases the mutation rate to accelerate adaptation to stressful environments. In this study, we first confirmed that 24-h treatment with areca nut extracts (ANE; a major component of BQ) at doses over 40 microg/ml induced mutations at the hypoxanthine phosphoribisyltransferase (HPRT) locus in human keratinocytes (HaCaT cells). We then investigated whether the stress of long-term exposure to sublethal doses of ANE (0, 5 and 20 microg/ml for 35 passages) could enhance genetic damage to HaCaT cells. Compared to cells exposed to 0 or 5 microg/ml ANE, cells exposed to 20 microg/ml ANE were slightly but significantly more resistant to a 72-h treatment with ANE and its major ingredients, arecoline and arecaidine, but did not develop cross-resistance to other BQ ingredients or alcohol. The cells that received 20 microg/ml ANE for 35 passages also had a significantly increased mutation frequency at the HPRT locus and an increased frequency in the appearance of micronuclei compared to lower doses. Moreover, increased intracellular levels of reactive oxygen species and 8-hydroxyguanosine in cells exposed to 20 microg/ml ANE suggested that long-term ANE exposure results in the accumulation of oxidative damage. However, cells subjected to long-term treatment of 20 microg/ml ANE contained higher levels of glutathione than unexposed cells. Therefore, after long-term exposure to sublethal doses of ANE, intracellular antioxidative activity may also be enhanced in response to increased oxidative stress. These results suggest that stress caused by long-term ANE exposure enhances oxidative stress and genetic damage in human keratinocytes.     

Anemonin attenuates osteoarthritis progression through inhibiting the activation of IL‐1β/NF‐κB pathway

The osteoarthritis (OA) progression is now considered to be related to inflammation. Anemonin (ANE) is a small natural molecule extracted from various kinds of Chinese traditional herbs and has been shown to inhibiting inflammation response. In this study, we examined whether ANE could attenuate the progression of OA via suppression of IL‐1β/NF‐κB pathway activation. Destabilization of the medial meniscus (DMM) was performed in 10‐week‐old male C57BL/6J mice. ANE was then intra‐articularly injected into joint capsule for 8 and 12 weeks. Human articular chondrocytes and cartilage explants challenged with interleukin‐1β (IL‐1β) were treated with ANE. We found that ANE delayed articular cartilage degeneration in vitro and in vivo. In particular, proteoglycan loss and chondrocyte hypertrophy were significantly decreased in ANE ‐treated mice compared with vehicle‐treated mice. ANE decreased the expressions of matrix metalloproteinase‐13 (MMP13), A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), collagen X (Col X) while increasing Aggrecan level in murine with DMM surgery. ANE treatment also attenuated proteoglycan loss in human cartilage explants treated with IL‐1β ex vivo. ANE is a potent protective molecule for OA; it delays OA progression by suppressing ECM loss and chondrocyte hypertrophy partially by suppressing IL‐1β/NF‐κB pathway activation.     

RNA-binding protein RBM14 regulates dissociation and association of non-homologous end joining proteins

Defects in the DNA damage response (DDR) are associated with multiple diseases, including cancers and neurodegenerative disorders. Emerging evidence indicates involvement of RNA-binding proteins (RBPs) in DDR. However, functions of RBPs in the DDR pathway remain elusive. We have shown previously that the RNA-binding protein RBM14 is required for non-homologous end joining (NHEJ). Here we show that RBM14 is required for efficient recruitment of XRCC4 and XLF to chromatin and the release of KU proteins from chromatin upon DNA damage. Failure of this process leads to accumulation of double-strand breaks (DSBs) in cells. Thus RBM14 plays crucial role in regulation of NHEJ upon DNA damage.     

Positive correlation between the expression of X-chromosome RBM genes (RBMX, RBM3, RBM10) and the proapoptotic Bax gene in human breast cancer

In a recent report, it has been postulated that the ubiquitous RBM proteins might constitute a novel family of apoptosis modulators. We measured the expression of the X-chromosome RBM genes (RBMX, RBM3, and RBM10) in 122 breast cancers by means of differential RT-PCR. Using the same method, we also studied the expression of the apoptosis-related genes Bcl-2 and Bax. Markers of hormone dependence (estrogen and progesterone receptors), proliferation (Ki67 and DNA-ploidy), angiogenesis (VEGF and CD105), as well as oncogene (c-erb-B2), and tumor suppressor gene (p53) expression were also analyzed. The expression of all X-chromosome RBM genes was significantly associated with the expression of the proapoptotic Bax gene (RBMX, P=0.039; RBM3, P<0.001; RBM10 large variant, P<0.001; RBM10 small variant, P<0.001). Furthermore, the expression of both RBM10 variants was significantly associated with the expression of the VEGF gene (large variant, P=0.004; small variant, P=0.003). We also found an association of borderline significance (P=0.05) between the expression of RBM3, the large variant of RBM10 and wild-type p53. Expression of the small RBM10 variant, finally, was associated with high proliferation of the tumors (Ki67>or=20%; P=0.037). The expression of both RBM10 variants seems to be interdependent to a significant degree (r=0.26, P=0.006). From these results, it seems that the X-chromosome, through its RBM genes, plays a formerly unknown role in the regulation of programmed cell death (apoptosis) in breast cancer.     

Autophagy induction by a natural ingredient of areca nut

We recently identified an autophagy-inducing areca nut ingredient (AIAI) in the partially purified 30-100 kDa fraction of areca nut extract (ANE), designated as ANE 30-100K. Before disintegration, most ANE 30-100K-treated cells exhibit rounding morphology, cytoplasmic clearance, and nuclear shrinkage, distinct from arecoline- and cisplatin-induced cellular apoptosis. This unique death pattern is verified to be autophagy by LC3-I cleavage, acidic vesicles, and autophagic vacuoles. As analyzed by Molish's Test, Selinowaff's Test, and thin-layer chromatography, most of the ANE 30-100K constituents are carbohydrates, whereas the protein content of this fraction is less than 1% as assessed by protein assay reagent. The cytotoxicity of ANE 30-100K is further shown to be sensitive to cellulase and proteinase K digestion suggesting AIAI in ANE 30-100K to be a proteoglycan (or glycoprotein). Thus, although ANE contains apoptosis-inducing ingredients such as arecoline, it predominantly triggers autophagic cell death by this natural AIAI.