Ablation of the dystrophin Dp71f alternative C-terminal variant increases sarcoma tumour cell aggressiveness.

Alterations in Dp71 expression, the most ubiquitous dystrophin isoform, have been associated with patient survival across tumours. Intriguingly, in certain malignancies, Dp71 acts as a tumour suppressor, while manifesting oncogenic properties in others. This diversity could be explained by the expression of two Dp71 splice variants encoding proteins with distinct C-termini, each with specific properties. Expression of these variants has impeded the exploration of their unique roles. Using CRISPR/Cas9, we ablated the Dp71f variant with the alternative C-terminus in a sarcoma cell line not expressing the canonical C-terminal variant, and conducted molecular (RNAseq) and functional characterisation of the knockout cells. Dp71f ablation induced major transcriptomic alterations, particularly affecting the expression of genes involved in calcium signalling and ECM-receptor interaction pathways. The genome-scale metabolic analysis identified significant downregulation of glucose transport via membrane vesicle reaction (GLCter) and downregulated glycolysis/gluconeogenesis pathway. Functionally, these molecular changes corresponded with, increased calcium responses, cell adhesion, proliferation, survival under serum starvation and chemotherapeutic resistance. Knockout cells showed reduced GLUT1 protein expression, survival without attachment and their migration and invasion in vitro and in vivo were unaltered, despite increased matrix metalloproteinases release. Our findings emphasise the importance of alternative splicing of dystrophin transcripts and underscore the role of the Dp71f variant, which appears to govern distinct cellular processes frequently dysregulated in tumour cells. The loss of this regulatory mechanism promotes sarcoma cell survival and treatment resistance. Thus, Dp71f is a target for future investigations exploring the intricate functions of specific DMD transcripts in physiology and across malignancies.

Downregulation of Dystrophin Expression Occurs across Diverse Tumors, Correlates with the Age of Onset, Staging and Reduced Survival of Patients.

Altered dystrophin expression was found in some tumors and recent studies identified a developmental onset of Duchenne muscular dystrophy (DMD). Given that embryogenesis and carcinogenesis share many mechanisms, we analyzed a broad spectrum of tumors to establish whether dystrophin alteration evokes related outcomes. Transcriptomic, proteomic, and mutation datasets from fifty tumor tissues and matching controls (10,894 samples) and 140 corresponding tumor cell lines were analyzed. Interestingly, dystrophin transcripts and protein expression were found widespread across healthy tissues and at housekeeping gene levels. In 80% of tumors, DMD expression was reduced due to transcriptional downregulation and not somatic mutations. The full-length transcript encoding Dp427 was decreased in 68% of tumors, while Dp71 variants showed variability of expression. Notably, low expression of dystrophins was associated with a more advanced stage, older age of onset, and reduced survival across different tumors. Hierarchical clustering analysis of DMD transcripts distinguished malignant from control tissues. Transcriptomes of primary tumors and tumor cell lines with low DMD expression showed enrichment of specific pathways in the differentially expressed genes. Pathways consistently identified: ECM-receptor interaction, calcium signaling, and PI3K-Akt are also altered in DMD muscle. Therefore, the importance of this largest known gene extends beyond its roles identified in DMD, and certainly into oncology.

Inflammation-Driven Secretion Potential Is Upregulated in Osteoarthritic Fibroblast-Like Synoviocytes.

Osteoarthritis (OA) is one of the most common joint pathologies and a major cause of disability among the population of developed countries. It manifests as a gradual degeneration of the cartilage and subchondral part of the bone, leading to joint damage. Recent studies indicate that not only the cells that make up the articular cartilage but also the synoviocytes, which build the membrane surrounding the joint, contribute to the development of OA. Therefore, the aim of the study was to determine the response to inflammatory factors of osteoarthritic synoviocytes and to identify proteins secreted by them that may influence the progression of OA. This study demonstrated that fibroblast-like synoviocytes of OA patients (FLS-OA) respond more strongly to pro-inflammatory stimulation than cells obtained from control patients (FLS). These changes were observed at the transcriptome level and subsequently confirmed by protein analysis. FLS-OA stimulated by pro-inflammatory factors [such as lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNFα) were shown to secrete significantly more chemokines (CXCL6, CXCL10, and CXCL16) and growth factors [angiopoietin-like protein 1 (ANGPTL1), fibroblast growth factor 5 (FGF5), and insulin-like growth factor 2 (IGF2)] than control cells. Moreover, the translation of proteolytic enzymes [matrix metalloprotease 3 (MMP3), cathepsin K (CTSK), and cathepsin S (CTSS)] by FLS-OA is increased under inflammatory conditions. Our data indicate that the FLS of OA patients are functionally altered, resulting in an enhanced response to the presence of pro-inflammatory factors in the environment, manifested by the increased production of the previously mentioned proteins, which may promote further disease progression.

Gene Expression Changes Induced by Exposure of RAW 264.7 Macrophages to Particulate Matter of Air Pollution: The Role of Endotoxins.

Despite the variable chemical and physical characteristics of particulate air pollutants, inflammation and oxidative stress have been identified as common mechanisms for cell damage and negative health influences. These effects are produced by organic components, especially by endotoxins. This study analyzed the gene expression profile after exposure of RAW 264.7 cells to the standard particulate matter (PM) material, NIST1648a, and PM with a reduced organic matter content, LAp120, in comparison to the effects of lipopolysaccharide (LPS). The selected parameters of cell viability, cell cycle progression, and metabolic and inflammatory activity were also investigated. Both forms of PM negatively influenced the parameters of cell activity. These results were generally reflected in the gene expression profile. Only NIST1648a, excluding LAp120, contained endotoxins and showed small but statistically significant pro-inflammatory activity. However, the gene expression profiling revealed strong pro-inflammatory cell activation induced by NIST1648a that was close to the effects of LPS. Changes in gene expression triggered by LAp120 were relatively small. The observed differences in the effects of NIST1648a and LAp120 were related to the content of organic matter in which bacterial endotoxins play an important role. However, other organic compounds and their interactions with other PM components also appear to be of significant importance.

Comparison of blood pressure values and expression of genes associated with hypertension in children before and after hematopoietic cell transplantation.

Hypertension is a well-known late effect of hematopoietic cell transplantation (HCT), but no markers predicting its development are known. Our aim was to assess short-term blood pressure (BP) values and expressions of hypertension-associated genes as possible markers of hypertension in children treated with HCT. We measured systolic blood pressure (SBP) and diastolic blood pressure (DBP), using both office procedure and ambulatory BP monitoring (ABPM) in children before HCT and after a median of 6 months after HCT. We compared the results with two control groups, one of healthy children and another of children with simple obesity. We also performed microarray analysis of hypertension-associated genes in patients treated with HCT and children with obesity. We found no significant differences in SBP and DBP in patients before and after HCT. We found significant differences in expressions of certain genes in patients treated with HCT compared with children with obesity. We concluded that BP values in short-term follow-up after HCT do not seem to be useful predictors of hypertension as a late effect of HCT. However, over expressions of certain hypertension-associated genes might be used as markers of hypertension as a late effect of HCT if this is confirmed in larger long-term studies.

Opposite regulation of piRNAs, rRNAs and miRNAs in the blood after subarachnoid hemorrhage.

Multiple classes of small RNAs (sRNAs) are expressed in the blood and are involved in the regulation of pivotal cellular processes. We aimed to elucidate the expression patterns and functional roles of sRNAs in the systemic response to intracranial aneurysm (IA) rupture. We used next-generation sequencing to analyze the expression of sRNAs in patients in the acute phase of IA rupture (first 72 h), in the chronic phase (3-15 months), and controls. The patterns of alterations in sRNA expression were analyzed in the context of clinically relevant information regarding the biological consequences of IA rupture. We identified 542 differentially expressed sRNAs (108 piRNAs, 99 rRNAs, 90 miRNAs, 43 scRNAs, 36 tRNAs, and 32 snoRNAs) among the studied groups with notable differences in upregulated and downregulated sRNAs between the groups and sRNAs categories. piRNAs and rRNAs showed a substantial decrease in RNA abundance that was sustained after IA rupture, whereas miRNAs were largely upregulated. Downregulated sRNA genes included piR-31080, piR-57947, 5S rRNA, LSU-rRNA, and SSU-rRNA s. Remarkable enrichment in the representation of transcription factor binding sites was revealed in genomic locations of the regulated sRNA. We found strong overrepresentation of glucocorticoid receptor, retinoid x receptor alpha, and estrogen receptor alpha binding sites at the locations of downregulated piRNAs, tRNAs, and rRNAs. This report, although preliminary and largely proof-of-concept, is the first to describe alterations in sRNAs abundance levels in response to IA rupture in humans. The obtained results indicate novel mechanisms that may constitute another level of control of the inflammatory response. KEY MESSAGES: A total of 542 sRNAs were differentially expressed after aneurysmal SAH comparing with controls piRNAs and rRNAs were upregulated and miRNAs were downregulated after IA rupture The regulated sRNA showed an enrichment in the representation of some transcription factor binding sites piRNAs, tRNAs, and rRNAs showed an overrepresentation for GR, RXRA, and ERALPHA binding sites.

Gastrointestinal peptides in children before and after hematopoietic stem cell transplantation.

BACKGROUND: Gastrointestinal tract function and it's integrity are controlled by a number of peptides whose secretion is influenced by severe inflammation. In stomach the main regulatory peptide is ghrelin. For upper small intestine cholecystokinin and lower small intestine glucagon-like peptide- 1 are secreted, while fibroblast growth factor-21 is secreted by several organs, including the liver, pancreas, and adipose tissue [12]. Hematopoietic stem cell transplantation causes serious mucosal damage, which can reflect on this peptides. METHODS: The aim of the study was to determine fasting plasma concentrations of ghrelin, cholecystokinin, glucagon- like peptide-1, and fibroblast growth factor-21, and their gene expressions, before and 6 months after hematopoietic stem cell transplantation.27 children were studied, control group included 26 healthy children. RESULTS: Acute graft versus host disease was diagnosed in 11 patients (41%, n = 27). Median pre-transplantation concentrations of gastrointestinal peptides, as well as their gene expressions, were significantly lower in studied group compared with the control group. Only median of fibroblast growth factor-21 concentration was near-significantly higher before stem cell transplantation than in the control group. The post-hematopoietic transplant results revealed significantly higher concentrations of the studied peptides (except fibroblast growth factor-21) and respective gene expressions as compare to pre transplant results. Median glucagone like peptide-1 concentrations were significantly decreased in patients with features of acute graft versus host disease. Moreover, negative correlation between glucagone like peptide-1 concentrations and acute graft versus host disease severity was found. CONCLUSIONS: Increased concentrations and gene expressions of gastrointestinal tract regulation peptides can be caused by stimulation of regeneration in the severe injured organ. Measurement of these parameters may be a useful method of assessment of severity of gastrointestinal tract complications of hematopoietic stem cell transplantation.

Genetic Profile and Clinical Implications of Hepatoblastoma and Neuroblastoma Coexistence in a Child.

The aim of the following case report is to provide a description of the coexistence of two independent tumors in a child. A 9-month-old male was referred to Department of Pediatric Oncology and Hematology with hepatic tumor present on ultrasound imaging and symptoms of enlarged abdominal circumference. Physical examination revealed a palpable epigastric mass and the imaging techniques showed a tumor of the left hepatic lobe measuring 11 × 6.5 × 8.9 cm with pancreas infiltration, distant metastases in both lungs and abnormal lesion in the left adrenal gland. Basing on histopathological examination, after a core-needle biopsy, hepatoblastoma (HBL) (mixed epithelial-mesenchymal subtype) was diagnosed. The α-fetoprotein level was 112 993 ng/ml. Elevated values of normetanephrine, 3-methoxytyramine as well as neuron-specific enolase were observed. Due to the clinical picture and diagnosis, the patient was qualified to preoperative chemotherapy according to the SIOPEL-3 protocol, followed by SIOPEL-4 protocol for the high-risk patients. After undergoing preoperative chemotherapy, imaging tests revealed regression of hepatic tumor and no focal pulmonary masses, while regression of adrenal gland mass was not completed. The patient was qualified for left hemihepatectomy with left adrenalectomy. Histopathological examination of liver specimen confirmed the HBL diagnosis. However, in left adrenal gland and paraaortic lymph nodes the residual neuroblastoma (NBL) cells were detected. Whole exome sequencing (WES) was utilized to identify disease-associated germline mutations. WES revealed a novel germline insertion variant in TWIST1 (p.Gly86dup), along with the potentially pathogenic non-synonymous variants in NF1 (p.Val2511Ile), RAF1 (p.Leu445Arg), and WHSC1 (p.Ser4Asn) genes. Currently, 6 months after completion of treatment according to the SIOPEL-4 protocol, the patient is in good general condition, without any signs, and symptoms of relapse of both neoplasms. The coexistence of two different primary childhood malignancies is rarely seen. So far, only one case of synchronous HBL and NBL has been reported. However, for the first time therapeutic process was successful. A specific signature of rare germline mutations can be proposed as a predisposing factor to synchronous HBL and NBL occurrence.

Genetic Signature of Acute Lymphoblastic Leukemia and Netherton Syndrome Co-incidence-First Report in the Literature.

The aim of the following case report is to provide a description of acute lymphoblastic leukemia (ALL) in a patient with Netherton syndrome (NS). A 15-year-old male with NS was referred with suspicion of acute leukemia. Severe anemia, leukocytosis, thrombocytopenia, and elevated CRP level were demonstrated in pre-hospital laboratory tests. Physical examination revealed generalized ichthyosiform erythroderma. ALL was diagnosed on the basis of bone marrow biopsy. The patient was initially classified as CNS3 status. No signals indicating fusion of BCR/ABL1, ETV6, and RUNX1 genes and MLL gene rearrangement were found in the cytogenetic analysis. The patient was qualified for chemotherapy and treated according to ALL IC-BFM 2009 protocol for high-risk ALL. During induction therapy, severe skin toxicity occurred (WHO grade III), which prompted the modification of treatment down to intermediate-risk strategy. In the course of reinduction therapy, severe chemotherapy-induced adverse drug reactions occurred, including progression of skin toxicity to WHO grade IV. The patient achieved complete remission. In view of life-threatening toxicities and the confirmed complete remission, intensive chemotherapy regimen was discontinued and maintenance treatment was started. Because of the baseline CNS3 status, the patient received cranial radiotherapy. Whole exome sequencing (WES) was used to identify disease-associated mutations. WES revealed two germline mutations: a novel premature termination variant in SPINK5 (p.Cys510*), along with a novel potentially pathogenic variant in NUP214 (p.Arg815Gln). Somatic mutations were known pathogenic variants of JAK2 (p.Arg683Gly), IL17RC (p.Ala303Thr), and potentially pathogenic non-synonymous variants of TTN (p.Gly1091Arg and p.Pro17245Leu), ACTN2 (p.Ile143Leu), TRPV3 (p.Arg729*), and COL7A1 (p.Glu2842fs) genes. Currently, the patient continues maintenance chemotherapy, with stable status of skin lesions and no features of ALL relapse. To our knowledge, this is the first report of ALL in a patient with NS. As has been presented, in such patients, optimal treatment according to the current protocols is extremely difficult. WES was used to confirm the diagnosis of Ph-like ALL in our patient. The detection of JAK2 gene mutation offers the possibility of therapy personalization. A specific signature of rare germline variants and somatic mutations can be proposed as a factor predisposing to the co-incidence of ALL and NS.

The topology of the lymphotoxin ß receptor that accumulates upon endolysosomal dysfunction dictates the NF-κB signaling outcome.

Cytokine receptors, such as tumor necrosis factor receptor I (TNFRI, also known as TNFRSF1A) and lymphotoxin ß receptor (LTßR), activate inflammatory nuclear factor (NF)-κB signaling upon stimulation. We have previously demonstrated that depletion of ESCRT components leads to endosomal accumulation of TNFRI and LTßR, and their ligand-independent signaling to NF-κB. Here, we studied whether other perturbations of the endolysosomal system could trigger intracellular accumulation and signaling of ligand-free LTßR. While depletion of the CORVET components had no effect, knockdown of Rab7a or HOPS components, or pharmacological inhibition of lysosomal degradation, caused endosomal accumulation of LTßR and increased its interaction with the TRAF2 and TRAF3 signaling adaptors. However, the NF-κB pathway was not activated under these conditions. We found that knockdown of Rab7a or HOPS components led to sequestration of LTßR in intraluminal vesicles of endosomes, thus precluding NF-κB signaling. This was in contrast to the LTßR localization on the outer endosomal membrane that was seen after ESCRT depletion and was permissive for signaling. We propose that the inflammatory response induced by intracellular accumulation of endocytosed cytokine receptors critically depends on the precise receptor topology within endosomal compartments.

Transcriptional signatures of steroid hormones in the striatal neurons and astrocytes.

BACKGROUND: The mechanisms of steroids actions in the brain mainly involve the binding and nuclear translocation of specific cytoplasmic receptors. These receptors can act as transcription factors and regulate gene expression. However, steroid-dependent transcriptional regulation in different types of neural cells is not yet fully understood. The aim of this study was to evaluate and compare transcriptional alterations induced by various steroid receptor agonists in primary cultures of astrocytes and neurons from mouse brain. RESULTS: We utilized whole-genome microarrays (Illumina Mouse WG-6) and quantitative PCR analyses to measure mRNA abundance levels. To stimulate gene expression we treated neuronal and astroglial cultures with dexamethasone (100 nM), aldosterone (200 nM), progesterone (200 nM), 5α-dihydrotestosterone (200 nM) and ß-Estradiol (200 nM) for 4 h. Neurons were found to exhibit higher levels of expression of mineralocorticoid receptor, progesterone receptor and estrogen receptor 2 than astrocytes. However, higher mRNA level of glucocorticoid receptor mRNA was observed in astrocytes. We identified 956 genes regulated by steroids. In astrocytes we found 381 genes altered by dexamethasone and 19 altered by aldosterone. Functional classification of the regulated genes indicated their putative involvement in multiple aspects of cell metabolism (up-regulated Slc2a1, Pdk4 and Slc45a3) and the inflammatory response (down-regulated Ccl3, Il1b and Tnf). Progesterone, dihydrotestosterone and estradiol did not change gene expression in astrocytes. We found no significant changes in gene expression in neurons. CONCLUSIONS: The obtained results indicate that glial cells might be the primary targets of transcriptional action of steroids in the central nervous system. Substantial changes in gene expression driven by the glucocorticoid receptor imply an important role for the hypothalamic-pituitary-adrenal axis in the hormone-dependent regulation of brain physiology. This is an in vitro study. Hence, the model may not accurately reflect all the effects of steroids on gene expression in neurons in vivo.

Mechanisms of Attenuation of Pulmonary V'O2 Slow Component in Humans after Prolonged Endurance Training.

In this study we have examined the effect of prolonged endurance training program on the pulmonary oxygen uptake (V'O2) kinetics during heavy-intensity cycling-exercise and its impact on maximal cycling and running performance. Twelve healthy, physically active men (mean±SD: age 22.33±1.44 years, V'O2peak 3198±458 mL ∙ min-1) performed an endurance training composed mainly of moderate-intensity cycling, lasting 20 weeks. Training resulted in a decrease (by ~5%, P = 0.027) in V'O2 during prior low-intensity exercise (20 W) and in shortening of τp of the V'O2 on-kinetics (30.1±5.9 s vs. 25.4±1.5 s, P = 0.007) during subsequent heavy-intensity cycling. This was accompanied by a decrease of the slow component of V'O2 on-kinetics by 49% (P = 0.001) and a decrease in the end-exercise V'O2 by ~5% (P = 0.005). An increase (P = 0.02) in the vascular endothelial growth factor receptor 2 mRNA level and a tendency (P = 0.06) to higher capillary-to-fiber ratio in the vastus lateralis muscle were found after training (n = 11). No significant effect of training on the V'O2peak was found (P = 0.12). However, the power output reached at the lactate threshold increased by 19% (P = 0.01). The power output obtained at the V'O2peak increased by 14% (P = 0.003) and the time of 1,500-m performance decreased by 5% (P = 0.001). Computer modeling of the skeletal muscle bioenergetic system suggests that the training-induced decrease in the slow component of V'O2 on-kinetics found in the present study is mainly caused by two factors: an intensification of the each-step activation (ESA) of oxidative phosphorylation (OXPHOS) complexes after training and decrease in the ''additional" ATP usage rising gradually during heavy-intensity exercise.

Expression profiling of genes modulated by minocycline in a rat model of neuropathic pain.

BACKGROUND: The molecular mechanisms underlying neuropathic pain are constantly being studied to create new opportunities to prevent or alleviate neuropathic pain. The aim of our study was to determine the gene expression changes induced by sciatic nerve chronic constriction injury (CCI) that are modulated by minocycline, which can effectively diminish neuropathic pain in animal studies. The genes associated with minocycline efficacy in neuropathic pain should provide insight into the etiology of neuropathic pain and identify novel therapeutic targets. RESULTS: We screened the ipsilateral dorsal part of the lumbar spinal cord of the rat CCI model for differentially expressed genes. Out of 22,500 studied transcripts, the abundance levels of 93 transcripts were altered following sciatic nerve ligation. Percentage analysis revealed that 54 transcripts were not affected by the repeated administration of minocycline (30 mg/kg, i.p.), but the levels of 39 transcripts were modulated following minocycline treatment. We then selected two gene expression patterns, B1 and B2. The first transcription pattern, B1, consisted of 10 mRNA transcripts that increased in abundance after injury, and minocycline treatment reversed or inhibited the effect of the injury; the B2 transcription pattern consisted of 7 mRNA transcripts whose abundance decreased following sciatic nerve ligation, and minocycline treatment reversed the effect of the injury. Based on the literature, we selected seven genes for further analysis: Cd40, Clec7a, Apobec3b, Slc7a7, and Fam22f from pattern B1 and Rwdd3 and Gimap5 from pattern B2. Additionally, these genes were analyzed using quantitative PCR to determine the transcriptional changes strongly related to the development of neuropathic pain; the ipsilateral DRGs (L4-L6) were also collected and analyzed in these rats using qPCR. CONCLUSION: In this work, we confirmed gene expression alterations previously identified by microarray analysis in the spinal cord and analyzed the expression of selected genes in the DRG. Moreover, we reviewed the literature to illustrate the relevance of these findings for neuropathic pain development and therapy. Further studies are needed to elucidate the roles of the individual genes in neuropathic pain and to determine the therapeutic role of minocycline in the rat neuropathic pain model.

Gene expression profiling of blood in ruptured intracranial aneurysms: in search of biomarkers.

The molecular mechanisms underlying the systemic response to subarachnoid hemorrhage (SAH) from ruptured intracranial aneurysms (RAs) are not fully understood. We investigated whether the analysis of gene expression in peripheral blood could provide clinically relevant information regarding the biologic consequences of SAH. Transcriptomics were performed using Illumina HumanHT-12v4 microarrays for 43 RA patients and 18 controls (C). Differentially expressed transcripts were analyzed for overrepresented functional groups and blood cell type-specific gene expression. The set of differentially expressed transcripts was validated using quantitative polymerase chain reaction in an independent group of subjects (15 RA patients and 14 C). There were 135 differentially expressed genes (false discovery rate 1%, absolute fold change 1.7): the abundant levels of 78 mRNAs increased and 57 mRNAs decreased. Among RA patients, transcripts specific to T lymphocyte subpopulations were downregulated, whereas those related to monocytes and neutrophils were upregulated. Expression profiles of a set of 16 genes and lymphocyte-to-monocyte-and-neutrophil gene expression ratios distinguished RA patients from C. These results indicate that SAH from RAs strongly influences the transcription profiles of blood cells. A specific pattern of these changes suggests suppression in lymphocyte response and enhancements in monocyte and neutrophil activities. This is probably related to the immunodepression observed in SAH.

Endurance training decreases the non-linearity in the oxygen uptake-power output relationship in humans.

In this study, we hypothesized that 5 weeks of cycling endurance training can decrease the magnitude of the non-proportional increase in oxygen uptake (V(O(2))) to power output relationship (V(O(2)) 'excess') at exercise intensities exceeding the lactate threshold (LT). Ten untrained, physically active men performed a bout of incremental cycling exercise until exhaustion before and after training. The mitochondrial DNA copy number, myosin heavy chain composition and content of uncoupling protein 3 and sarcoplasmic reticulum Ca(2+)-ATPases (SERCAs) were analysed in muscle biopsies taken from vastus lateralis before and after training. The training resulted in an enhancement of the power-generating capabilities at maximal oxygen uptake (V(O(2)max)) by ∼7% (P = 0.002) despite there being no changes in V(O(2)max) (P = 0.49). This effect was due to a considerable reduction in the magnitude of the V(O(2)) 'excess' (P < 0.05) above the LT. A decrease in plasma ammonia concentration was found during exercise after training (P < 0.05). A downregulation of SERCA2 in vastus lateralis (P = 0.006) was observed after training. No changes in myosin heavy chain composition, selected electron transport chain proteins, uncoupling protein 3 or the mitochondrial DNA copy number (P > 0.05) were found after training. We conclude that the training-induced increase in power-generating capabilities at V(O(2)max) was due to attenuation of the V(O(2)) 'excess' above the LT. This adaptive response seems to be related to the improvement of muscle metabolic stability, as judged by a lowering of plasma ammonia concentration. The enhancement of muscle metabolic stability after training could be caused by a decrease in ATP usage at a given power output owing to downregulation of SERCA2 pumps.

Identification of interleukin-1 and interleukin-6-responsive genes in human monocyte-derived macrophages using microarrays.

The transcriptome profile of human monocyte-derived macrophages stimulated in vitro by low doses of IL-1 or IL-6 was analyzed by microarrays (Affymetrix, HG-U133A) in 5 independent experiments. Out of 4886 probe sets consistently detected in all 5 array replicates we found approximately 300 genes (FDR<5%) modulated by IL-1 and/or IL-6, among which 34 may be regarded as novel cytokine-responsive macrophage genes of various function. Detailed analysis indicates that cytokine-responsive genes include 125 transcripts significantly up-regulated by IL-1 and only 39 transcripts up-regulated by IL-6, whereas the number of down-regulated transcripts is lower and almost equal for both cytokines. These data indicate that, in comparison to liver cells, IL-1 is more potent than IL-6 in modulating gene expression of human macrophages. Hierarchical clustering analysis of these transcripts yielded 7 separate gene clusters. The most abundant group contains genes strongly activated by IL-1 alone and coding for chemokines, cytokines and their receptors, the components of intracellular signaling as well as transcription factors from NF-kB family. In order to validate the results obtained by microarray analysis the expression of 5 genes from various clusters was determined by quantitative RT-PCR. Moreover, the putative promoter regions of all cytokine-responsive genes were subjected to the in silico identification of transcription factor binding sites (TFBS). We found that TFBS corresponding to RelA/NF-kB is the most strongly over-represented group and we demonstrated involvement of NF-kB in the expression of selected genes.