The positive regulatory machinery in the microRNA (miRNA) processing pathway is relatively well characterized, but negative regulation of the pathway is largely unknown. Here we show that a complex of nuclear factor 90 (NF90) and NF45 proteins functions as a negative regulator in miRNA biogenesis. Primary miRNA (pri-miRNA) processing into precursor miRNA (pre-miRNA) was inhibited by overexpression of the NF90 and NF45 proteins, and considerable amounts of pri-miRNAs accumulated in cells coexpressing NF90 and NF45. Treatment of cells overexpressing NF90 and NF45 with an RNA polymerase II inhibitor, ␣-amanitin, did not reduce the amounts of pri-miRNAs, suggesting that the accumulation of pri-miRNAs is not due to transcriptional activation. In addition, the NF90 and NF45 complex was not found to interact with the Microprocessor complex, which is a processing factor of primiRNAs, but was found to bind endogenous pri-miRNAs. NF90-NF45 exhibited higher binding activity for pri-let-7a than pri-miR-21. Of note, depletion of NF90 caused a reduction of pri-let-7a and an increase of mature let-7a miRNA, which has a potent antiproliferative activity, and caused growth suppression of transformed cells. These findings suggest that the association of the NF90-NF45 complex with pri-miRNAs impairs access of the Microprocessor complex to the pri-miRNAs, resulting in a reduction of mature miRNA production.MicroRNAs (miRNAs) constitute a class of noncoding small RNAs that function as repressors for eukaryotic gene regulation by binding to the 3Ј untranslated regions of target mRNAs (2). This binding causes mRNA cleavage or translational inhibition of the mRNA, depending upon the degree of complementarity. The lengths of miRNAs are 21 to 23 nucleotides (nt), and over 500 miRNAs have been discovered in mammals. miRNAs regulate the expression of a large number of genes (38) that are involved in cell proliferation, apoptosis, hematopoietic differentiation, viral infection, and tumorigenesis (4,5,7,22,26,32,39,45).In mammals, miRNA genes are transcribed by RNA polymerase II as primary miRNAs (pri-miRNAs) (36). These primiRNAs are processed into precursor miRNAs (pre-miRNAs) by the Microprocessor complex (8,13,20,31,33). Another complex comprised of exportin-5 and RanGTP transports the pre-miRNAs from the nucleus to the cytoplasm (3, 40, 58). In the cytoplasm, Dicer, a cytoplasmic RNase III enzyme, cleaves the pre-miRNAs to approximate 22-nt mature miRNA duplexes with 2-nt 3Ј overhangs (14,24,28). One strand of the duplex is incorporated into the RNA-induced silencing complex (12,19,29,41,51). The single strand of RNA guides the RNA-induced silencing complex to the target mRNA with sequence complementarity, which leads either to mRNA cleavage or to translational repression (12,24,41,44).The Microprocessor complex, which cleaves pri-miRNA to pre-miRNA during miRNA biogenesis, is comprised of a nuclear RNase III enzyme, Drosha, and its cofactor, DGCR8 (8,13,20). In addition to the Microprocessor complex, excessively expressed Drosha forms ...

Animals sense various ranges of temperatures by cutaneous thermal stimuli. Transient receptor potential vanilloid 4 (TRPV4) is a cation channel activated at a warm temperature (over 30°C) in exogenously expressed cells. We found in the present study that TRPV4 is essential in thermal hyperalgesia at a warm temperature in vivo. TRPV4؊/؊ and TRPV4؉/؉ mice exhibited the same latency of escape from 35-50°C hotplates. Neuronal activity in the femoral nerve, however, revealed that the number and activity level of neurons decreased in response to a warm temperature in TRPV4؊/؊ mice. TRPV4؊/؊ mice displayed a significantly longer latency to escape from the plates at 35-45°C when hyperalgesia was induced by carrageenan without changes in foot volumes. TRPV4 therefore determines the sensitivity rather than the threshold of painful heat detection and plays an essential role in thermal hyperalgesia.Sensation of warm to hot temperatures is an essential afferent neural activity in animals. An insight into the molecular basis of this sensation has been obtained by discovery of a capsaicin receptor, transient receptor potential vanilloid 1 (TRPV1) 1 (1). TRPV1 encodes a nonselective cation channel protein originally identified as the receptor for capsaicin, the principal pungent constituent in hot peppers. In addition to capsaicin and related vanilloid compounds, this channel is activated by acidic pH or by painfully hot temperatures. Responses to noxious heat are diminished in mice lacking TRPV1 (2). Detectable responses to hot temperatures (42-52°C) are absent in sensory neurons cultured from these animals. Therefore, TRPV1 plays an essential role in the detection of hot temperature. However, a significant component of heat responsiveness remains in TRPV1-knock-out mice and in skin-nerve preparations explanted from them (2, 3). These findings indicate the existence of TRPV1-independent mechanisms of heat detection.The mechanisms by which mammals detect innocuous warm temperatures are even less well understood than the mechanism underlying noxious thermosensation. A subset of sensory nerve fibers responsive to heat ranging from 30 to 42°C has been identified in vivo in a number of mammalian species (4, 5), although the heat transduction mechanisms accounting for such responsiveness have not been clarified. Recently, it has been reported that two TRPV1-related ion channel proteins, TRPV3 and TRPV4, can be activated by mild temperature elevations exceeding ϳ30 -35°C (6, 7). TRPV3 and TRPV4 are located in cultured keratinocytes and play an essential role in the detection of warm temperatures and hypoosmolarity in vitro (8).We previously found that TRPV4 might be a mediator of sensory neuron responsiveness to hypoosmolarity as well as a contributor to mechanical nociception (9 -11) in vivo by using mice lacking TRPV4. However, the role of TRPV4 in the detection of warm temperatures in vivo remains obscure.The mice lacking TRPV1 showed impairment in detection of chemically induced inflammatory pain when tested on hotplates over 50°...

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The concentration of gamma-aminobutyric acid (GABA) and the activity of glutamate decarboxylase (GAD) in rat and human pancreas were measured by sensitive assay methods. The GABA concentration in rat pancreas was 2.51 millimoles per kilogram (dry weight) and GAD activity was 2.58 mmoles per kilogram per hour. The GABA concentration and GAD activity in rat Langerhans' islets were 18.9 mmole kg-1 and 66.7 mmole kg-1 hour-1, whereas those in the exocrine acini were 1.97 mmole kg-1 and 4.67 mmole kg-1 hour-1, respectively. In an insulinoma region of human pancreas the GABA concentration was 25.5 mmole kg-1 and the GAD activity was 138.2 mmole kg-1 hour-1, but in the surrounding nontumor region these values were only 2.81 mmole kg-1 and 2.01 mmole kg-1 hour-1, respectively, similar to the values in normal rat pancreas.

These results suggest an association between chronic, life-threatening stress and the worsening of metabolic control in patients with diabetes mellitus.