RNA polymerase II transcribes most eukaryotic genes. Its catalytic subunit was tagged with green fluorescent protein and expressed in Chinese hamster cells bearing a mutation in the same subunit; it complemented the defect and so was functional. Photobleaching revealed two kinetic fractions of polymerase in living nuclei: ∼75% moved rapidly, but ∼25% was transiently immobile (association t1/2 ≈ 20 min) and transcriptionally active, as incubation with 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole eliminated it. No immobile but inactive fraction was detected, providing little support for the existence of a stable holoenzyme, or the slow stepwise assembly of a preinitiation complex on promoters or the nuclear substructure. Actinomycin D decreased the rapidly moving fraction, suggesting that engaged polymerases stall at intercalated molecules while others initiate. When wild-type cells containing only the endogenous enzyme were incubated with [3H]uridine, nascent transcripts became saturated with tritium with similar kinetics (t1/2 ≈ 14 min). These data are consistent with a polymerase being mobile for one half to five sixths of a transcription cycle, and rapid assembly into the preinitiation complex. Then, most expressed transcription units would spend significant times unassociated with engaged polymerases.

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Glioblastoma multiforme (GBM) is the most frequent and incurable type of brain tumor of adults. Hypoxia has been shown to direct GBM toward a more aggressive and malignant state. Here we show that hypoxia increases Notch1 activation, which in turn induces the expression of transient receptor potential 6 (TRPC6) in primary samples and cell lines derived from GBM. TRPC6 is required for the development of the aggressive phenotype because knockdown of TRPC6 expression inhibits glioma growth, invasion, and angiogenesis. Functionally, TRPC6 causes a sustained elevation of intracellular calcium that is coupled to the activation of the calcineurin-nuclear factor of activated T-cell (NFAT) pathway. Pharmacologic inhibition of the calcineurin-NFAT pathway substantially reduces the development of the malignant GBM phenotypes under hypoxia. Clinically, expression of TRPC6 was elevated in GBM specimens in comparison with normal tissues. Collectively, our studies indicate that TRPC6 is a key mediator of tumor growth of GBM in vitro and in vivo and that TRPC6 may be a promising therapeutic target in the treatment of human GBM.

Proteasomes are the multi-subunit protease thought to play a key role in the generation of peptides presented by major histocompatibility complex (MHC) class I molecules. When cells are stimulated with interferon y, two MHC-encoded subunits, low molecular mass polypeptide (LMP) 2 and LMP7, and the MECL1 subunit encoded outside the MHC are incorporated into the proteasomal complex, presumably by displacing the housekeeping subunits designated Y, X, and Z, respectively. These changes in the subunit composition appear to facilitate class I-mediated antigen presentation, presumably by altering the cleavage specificities of the proteasome. Here we show that the mouse gene encoding the Z subunit (Psmb7) maps to the paracentromeric region of chromosome 2. Inspection of the mouse loci adjacent to the Psmb7 locus provides evidence that the paracentromeric region of chromosome 2 and the MHC region on chromosome 17 most likely arose as a result of a duplication that took place at an early stage of vertebrate evolution. The traces of this duplication are also evident in the homologous human chromosome regions (6p21.3 and 9q33-q34). These observations have implications in understanding the genomic organization of the present-day MHC and offer insights into the origin of the MHC. Accumulated evidence indicates that the proteasome plays a key role in the generation of cytosolic peptides presented by major histocompatibility complex (MHC) class I molecules (reviewed in refs.

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To clarify the lower urinary tract function in mice, we compared bladder and urethral activity between rats and mice with or without spinal cord injury (SCI). Female Sprague-Dawley rats and C57BL/6N mice were divided into five groups:1) spinal intact (SI) rats,2) SI mice,3) pudendal nerve transection (PNT) SI mice,4) spinal cord injury (SCI) rats, and 5) SCI mice. Continuous cystometry (CMG) and external urethral sphincter (EUS)-electromyogram (EMG) analyses were conducted under an awake, restrained condition. During voiding bladder contractions, SI animals exhibited EUS bursting with alternating active and silent periods, which, in rats but not mice, coincided with small-amplitude intravesical pressure oscillations in CMG recordings. In SI mice with bursting-like EUS activity, the duration of active periods was significantly shorter by 46% (32 ± 5 ms) compared with SI rats (59 ± 9 ms). In PNT-SI mice, there were no significant differences in any of cystometric parameters compared with SI mice. In SCI rats, fluid elimination from the urethra and the EUS bursting occurred during small-amplitude intravesical pressure oscillations. However, SCI mice did not exhibit clear EUS bursting activity or intravesical pressure oscillations but rather exhibited intermittent voiding with slow large-amplitude reductions in intravesical pressure, which occurred during periods of reduced EUS activity. These results indicate that EUS pumping activity is essential for generating efficient voiding in rats with or without spinal cord injury. However, EUS bursting activity is not required for efficient voiding in SI mice and does not reemerge in SCI mice in which inefficient voiding occurs during periods of reduced tonic EUS activity.

The regulation of memory CD4(+) helper T (Th) cell function, such as polarized cytokine production, remains unclear. Here we show that memory T helper 2 (Th2) cells are divided into four subpopulations by CD62L and CXCR3 expression. All four subpopulations produced interleukin-4 (IL-4) and IL-13, whereas only the CD62L(lo)CXCR3(lo) population produced IL-5 accompanied by increased H3-K4 methylation at the Il5 gene locus. The transcription factor Eomesodermin (encoded by Eomes) was highly expressed in memory Th2 cells, whereas its expression was selectively downregulated in the IL-5-producing cells. Il5 expression was enhanced in Eomes-deficient cells, and Eomesodermin was shown to interact with the transcription factor GATA3, preventing GATA3 binding to the Il5 promoter. Memory Th2 cell-dependent airway inflammation was attenuated in the absence of the CD62L(lo)CXCR3(lo) population but was enhanced by Eomes-deficient memory Th2 cells. Thus, IL-5 production in memory Th2 cells is regulated by Eomesodermin via the inhibition of GATA3 activity.