Perceptual effects and efficacy of intermittent or continuous blood flow restriction resistance training.

Blood flow restriction (BFR) exercise may be an alternative form of resistance training; however, a side of effect of BFR resistance exercise is acute muscle pain. Typically, BFR exercise studies restrict blood flow with a cuff continuously during the exercise bout, including rest periods. However, others have used intermittent BFR where the cuff is inflated only during sets. We performed two studies to compare intermittent and continuous BFR exercise. In study one, eleven subjects randomly proceeded through three treatments of unilateral leg extensions to failure: (i) continuous BFR, (ii) intermittent BFR and (iii) control (exercise without BFR). Pain measurements were taken immediately after each set. In study two, subjects (n = 32) underwent a 5-week resistance training programme after random assignment to one of the three conditions. Lean mass and strength were assessed at baseline and after training. Continuous BFR resulted in significantly greater pain than intermittent BFR or control. Both BFR conditions resulted in significantly fewer repetitions to failure than control. This suggests that an acute bout of intermittent BFR exercise may produce as much muscle fatigue as an acute bout of continuous BFR exercise, but with less pain. With training, maximal knee extension (P = 0·033) and maximum knee flexion (P = 0·007) strength increased among all groups. There were no significant differences between groups in strength or lean mass. These results suggest that short-term low-load resistance training increases muscle strength to a similar extent as low-load resistance training without BFR.

The mutant plasmacytoma cell line S107 allows the identification of distinct pathways leading to NF-kappaB activation.

Studies on the mechanisms of inducible and constitutive activity of NF-kappaB transcription factors have been hampered by the lack of appropriate mutant cell lines. We have analyzed the defect in the murine S107 plasmacytoma cell line, which was previously found to lack both constitutive and inducible NF-kappaB activity. Our analysis shows that these cells bear a specific defect that interferes with NF-kappaB induction by many diverse stimuli, such as lipopolysaccharide, phorbol 12-myristate 13-acetate, UV light, x-rays, and H2O2. This does not however represent a general signal transduction defect, because AP-1 transcription factors are readily induced by the same stimuli. Phosphatase inhibitors such as okadaic acid as well as calyculin A can efficiently induce NF-kappaB in S107 cells via a pathway apparently insensitive to the radical scavenger pyrrolidine dithiocarbamate. Furthermore, MEKK1 a protein kinase supposedly induced by some of the above stimuli, is also capable of activating NF-kappaB. Interestingly, both the potent physiological inducer of NF-kappaB TNFalpha as well as endoplasmic reticulum overload can induce NF-kappaB via a PDTC sensitive pathway. In all cases, DNA-binding NF-kappaB complexes are comprised predominantly of p50-RelA heterodimers, and NF-kappaB activation results in the induction of transiently transfected or resident reporter genes. In summary, these results suggest that the pathways for many NF-kappaB-inducing stimuli converge at a specific junction, and this pivotal step is mutated in the S107 cell line. Yet there are alternative routes bypassing this critical step that also lead to NF-kappaB induction. These routes utilized by tumor necrosis factor alpha and endoplasmic reticulum overload are still intact in this cell line.

Induction of nuclear factor-kappa B during primary B cell differentiation.

We have investigated activation of nuclear factor-kappa B (NF-kappa B) in the process of primary B cell differentiation in vitro. In this system, NF-kappa B is strongly induced when B cells develop from the pre-B cell to the immature B cell stage. Unlike the typical NF-kappa B activation in response to exogenous stimuli, induction proceeds with a slow time course. NF-kappa B induction is only observed in B cells that undergo differentiation, not in Rag2-deficient cells. Nuclear DNA binding complexes predominantly comprise p50/RelA heterodimers and, to a lesser extent, c-Rel-containing dimers. The increase in NF-kappa B binding activity is accompanied by a slow and steady decrease in I kappa B beta protein levels. Interestingly, absolute RelA protein levels remain unaffected, whereas RelB and c-Rel synthesis is induced. The reason for preferential nuclear translocation of RelA complexes appears to be selective inhibition by the I kappa B beta protein. I kappa B beta can efficiently inhibit p50/RelA complexes, but has a much reduced ability to interfere with p50/c-Rel DNA binding both in vitro and in vivo. Interestingly, p50/RelB complexes are not at all targeted by I kappa B beta, and coimmunoprecipitation experiments show no evidence for an association of I kappa B beta and RelB in vivo. Consistent with these observations, I kappa B beta cotransfection can inhibit p50/RelA-mediated trans-activation, but barely affects p50/RelB mediated trans-activation.

Glutamate-dependent activation of NF-kappaB during mouse cerebellum development.

NF-kappaB and activator protein 1 (AP-1) are dimeric transcription factors involved in transcriptional regulation in many cells, including neurons. We have examined their activity during mouse cerebellum development, a postnatal process starting just after birth and completed by the fourth postnatal (PN) week. The activity of these factors was analyzed by binding of nuclear extracts to a synthetic oligonucleotide representing the kappaB site of human immunodeficiency virus or the AP-1 site of the urokinase promoter. NF-kappaB activity was observed from 7 PN, was restricted to the developing cerebellum, and was not observed in the early postnatal neocortex and hippocampus. On the other hand, AP-1 activity was not found in cerebellum but was present in both neocortex and hippocampus. Moreover, a kappaB-driven transgene was found to be increasingly expressed in the cerebellum from 5 PN to 10 PN but not in the adult. The regulation of NF-kappaB activation in mouse cerebellum was analyzed by intraperitoneal injection of glutamate receptor antagonists to 9 PN mice, which abolished NF-kappaB-binding activity, suggesting an endogenous loop of glutamate receptor activation. Glutamate receptor agonists, on the other hand, induced NF-kappaB nuclear translocation in the cerebellum of 5 PN mice, which is a stage in which NF-kappaB is not yet endogenously activated. This effect was specific for NF-kappaB and not observed for AP-1. In adult mice, NF-kappaB activity was absent in the cerebellum and was not induced by intraperitoneal injection of glutamate receptor agonists. These data show that NF-kappaB is specifically activated during cerebellum development and indicate an important role of glutamate receptors in this process.

RelB is a key player for both kappa B-dependent transcription and demethylation in B cells.

NF-kappa B was originally identified as a B cell-specific nuclear factor binding to the intronic kappa-light-chain enhancer element. It is found constitutively in the nucleus of mature B and plasma cells. In other cell types including pre-B cells, NF-kappa B is sequestered in the cytoplasm but can be induced by a variety of stimuli. In contrast to essentially all other mature B cells and plasma cell lines, the S107 plasmacytoma cell line lacks both constitutive and inducible kappa B-binding activity. A molecular characterization of the defect in these S107 cells suggests that the primary defect lies in the signal transduction pathway leading to NF-kappa B induction. Ectopic expression of RelB after stable transfection of these cells restores constitutive nuclear kappa B-binding activity. Moreover, kappa B-dependent transcription is also restored. Finally we demonstrate, that in contrast to parental S107 cells, the stable RelB transfectants have also regained the ability to specifically demethylate a transfected immunoglobulin kappa-locus. These data suggest that RelB is critically involved in both B cell-specific transcription and demethylation directed by the intronic kappa-enhancer element.

CD40, but not lipopolysaccharide and anti-IgM stimulation of primary B lymphocytes, leads to a persistent nuclear accumulation of RelB.

In this study we analyzed the effect of CD40 stimulation on the activity and nuclear appearance of Rel/nuclear factor kappaB (NF-kappaB) factors in primary murine B lymphocytes. We show that triggering of CD40 signaling pathway(s) by CD40 ligands expressed on L cells led to strong activation of an NF-kappaB-controlled beta-globin reporter gene in primary B lymphocytes from transgenic mice. Analyses of nuclear translocation of individual members of Rel proteins after CD40 induction of primary B cells showed a strong and long-lasting accumulation of RelB and, less pronounced, of c-Rel. LPS stimulation did not give rise to a persistent nuclear accumulation of RelB and c-Rel, whereas nuclear c-Rel, but not RelB, accumulated after B cell receptor stimulation. CD40 induced not only nuclear translocation but also de novo synthesis of RelB RNA and protein. S107 plasmacytoma cells, which express CD40 but are defective for the nuclear appearance of p50/p65-NF-kappaB, do not express RelB after CD40 stimulation. In S107 cells stably transfected with relB genes, stimulation of nuclear RelB translocation by CD40 was observed. These results indicate that stimulation of CD40 signaling pathways exerts a long-lasting stimulatory effect on both the transcription and nuclear translocation of RelB. Since LPS and anti-IgM were unable to activate RelB, CD40 appears to trigger a special program of gene expression involved in the proliferation and/or differentiation of B lymphocytes.

A role for nuclear NF-kappaB in B-cell-specific demethylation of the Igkappa locus.

The immunoglobulin kappa gene is specifically demethylated during B-cell maturation in a process which utilizes discrete cis-acting modules such as the intronic kappa enhancer element and the matrix attachment region (MAR). While any MAR sequence is sufficient for this reaction, mutation analysis indicates that tissue specificity is mediated by kappaB binding sequences within the kappa intronic enhancer. The plasmacytoma cell line S107 lacks kappaB binding activity and fails to demethylate the kappa locus. However, B-cell specific demethylation is restored by the introduction of an active kappaB binding protein gene relB. This represents the first demonstration of a trans-acting factor involved in cell-type-specific demethylation, and suggests that the same protein-DNA recognition system used for transcription may also contribute to the earlier developmental events that bring about activation of the kappa locus.

Lymphoid- and myeloid-specific activity of the PU.1 promoter is determined by the combinatorial action of octamer and ets transcription factors.

The putative oncogene PU.1/Spi-1 is a member of the ets-family of transcription factors normally expressed in a subset of hematopoietic cell types. Here we have characterized the role of the PU.1 promoter region for the cell-type specific expression. The proximal 120 bp are sufficient to mediate a high level of activity specifically in B cells and macrophages. Three important motifs could be identified within this region. Two of them, an ets binding site (EBS) and a variant octamer motif were most important for cell-type-specific promoter activity in B cells and macrophages. An additional Sp1 motif stimulates basal activity of this promoter element. The relative contribution to overall activity of octamer motif and EBS differs in B cells and macrophages. In B cells, both octamer motif and EBS combine to mediate high level activity, whereas in macrophages the EBS predominantly confers promoter activity. Both the Oct1 and Oct2 transcription factors, presumably in combination with a B-cell-restricted coactivator, are responsible for the activity of the variant octamer motif in B cells. Interestingly, the PU.1 transcription factor can functionally interact with the EBS in its own promoter, suggesting a positive feedback regulation.

Two distinct mechanisms contribute to the constitutive activation of RelB in lymphoid cells.

The kappa B-motif is an important regulatory element both for constitutive lymphoid-specific as well as ubiquitous inducible transcriptional activity. We have shown previously that different members of the NF-kappa B/Rel family of transcription factors are responsible for these distinct functions. Whereas the p65/RelA protein is involved in inducible kappa B-dependent transcription, RelB is associated with constitutive activity in lymphoid cells. Here we have addressed the question of how RelB is constitutively activated in lymphoid cells. We demonstrate that this is achieved by two different mechanisms. Expression of relB as determined by measurement of stable RNA and protein levels is significantly enhanced in lymphoid organs compared with non-lymphoid organs. However, these observed differences in absolute amounts of RNA and protein would not suffice to explain the dramatic differences that are apparent at the level of active DNA binding complexes in extracts from the respective organs. We have therefore analyzed the interaction of RelB complexes with the I kappa B-alpha inhibitor protein. Our results show that RelB-containing complexes present in lymphoid extracts are much less susceptible to inhibition by I kappa B-alpha than RelA- or RelB-containing complexes from non-lymphoid cells. This difference might be due to post-translational modifications of the RelB protein or interaction with a lymphoid-specific cofactor for RelB.