The proliferation and differentiation of cells of many lineages are regulated by secreted proteins known as cytokines. Cytokines exert their biological effect through binding to cell-surface receptors that are associated with one or more members of the JAK family of cytoplasmic tyrosine kinases. Cytokine-induced receptor dimerization leads to the activation of JAKs, rapid tyrosine-phosphorylation of the cytoplasmic domains, and subsequent recruitment of various signalling proteins, including members of the STAT family of transcription factors, to the receptor complex. Using the yeast two-hybrid system, we have now isolated a new SH2-domain-containing protein, JAB, which is a JAK-binding protein that interacts with the Jak2 tyrosine-kinase JH1 domain. JAB is structurally related to CIS, a cytokine-inducible SH2 protein. Interaction of JAB with Jak1, Jak2 or Jak3 markedly reduces their tyrosine-kinase activity and suppresses the tyrosine-phosphorylation and activation of STATs. JAB and CIS appear to function as negative regulators in the JAK signalling pathway.

We investigated the hemodynamic and hormonal responses to a short-term low-intensity resistance exercise (STLIRE) with the reduction of muscle blood flow. Eleven untrained men performed bilateral leg extension exercise under the reduction of muscle blood flow of the proximal end of both legs pressure-applied by a specially designed belt (a banding pressure of 1.3 times higher than resting systolic blood pressure, 160-180 mmHg), named as Kaatsu. The intensity of STLIRE was 20% of one repetition maximum. The subjects performed 30 repetitions, and after a 20-seconds rest, they performed three sets again until exhaustion. The superficial femoral arterial blood flow and hemodynamic parameters were measured by using the ultrasound and impedance cardiography. Serum concentrations of growth hormone (GH), vascular endothelial growth factor (VEGF), noradrenaline (NE), insulin-like growth factor (IGF)-1, ghrelin, and lactate were also measured. Under the conditions with Kaatsu, the arterial flow was reduced to about 30% of the control. STLIRE with Kaatsu significantly increased GH (0.11+/-0.03 to 8.6+/-1.1 ng/ml, P < 0.01), IGF-1 (210+/-40 to 236+/-56 ng/ml, P < 0.01), and VEGF (41+/-13 to 103+/-38 pg/ml, P < 0.05). The increase in GH was related to neither NE nor lactate, but the increase in VEGF was related to that in lactate (r = 0.57, P < 0.05). Ghrelin did not change during the exercise. The maximal heart rate (HR) and blood pressure (BP) in STLIRE with Kaatsu were higher than that without Kaatsu. Stroke volume (SV) was lower due to the decrease of the venous return by Kaatsu, but, total peripheral resistance (TPR) did not change significantly. These results suggest that STLIRE with Kaatsu significantly stimulates the exercise-induced GH, IGF, and VEGF responses with the reduction of cardiac preload during exercise, which may become a unique method for rehabilitation in patients with cardiovascular diseases.

Cellular proliferation, and differentiation of cells in response to extracellular signals, are controlled by the signal transduction pathway of Ras, Raf and MAP (mitogen-activated protein) kinase. The mechanisms that regulate this pathway are not well known. Here we describe two structurally similar tyrosine kinase substrates, Spred-1 and Spred-2. These two proteins contain a cysteine-rich domain related to Sprouty (the SPR domain) at the carboxy terminus. In Drosophila, Sprouty inhibits the signalling by receptors of fibroblast growth factor (FGF) and epidermal growth factor (EGF) by suppressing the MAP kinase pathway. Like Sprouty, Spred inhibited growth-factor-mediated activation of MAP kinase. The Ras-MAP kinase pathway is essential in the differentiation of neuronal cells and myocytes. Expression of a dominant negative form of Spred and Spred-antibody microinjection revealed that endogenous Spred regulates differentiation in these types of cells. Spred constitutively associated with Ras but did not prevent activation of Ras or membrane translocation of Raf. Instead, Spred inhibited the activation of MAP kinase by suppressing phosphorylation and activation of Raf. Spred may represent a class of proteins that modulate Ras-Raf interaction and MAP kinase signalling.

Previous studies using B16BL6-derived exosomes labelled with gLuc–lactadherin (gLuc-LA), a fusion protein of Gaussia luciferase (a reporter protein) and lactadherin (an exosome-tropic protein), showed that the exosomes quickly disappeared from the systemic circulation after intravenous injection in mice. In the present study, the mechanism of rapid clearance of intravenously injected B16BL6 exosomes was investigated. gLuc-LA-labelled exosomes were obtained from supernatant of B16BL6 cells after transfection with a plasmid DNA encoding gLuc-LA. Labelling was stable when the exosomes were incubated in serum. By using B16BL6 exosomes labelled with PKH26, a lipophilic fluorescent dye, it was demonstrated that PKH26-labelled B16BL6 exosomes were taken up by macrophages in the liver and spleen but not in the lung, while PKH26-labelled exosomes were taken up by the endothelial cells in the lung. Subsequently, gLuc-LA-labelled B16BL6 exosomes were injected into macrophage-depleted mice prepared by injection with clodronate-containing liposomes. The clearance of the intravenously injected B16BL6 exosomes from the blood circulation was much slower in macrophage-depleted mice than that in untreated mice. These results indicate that macrophages play important roles in the clearance of intravenously injected B16BL6 exosomes from the systemic circulation.

Circadian rhythms govern the behavior, physiology, and metabolism of living organisms. Recent studies have revealed the role of several genes in the clock mechanism both in Drosophila and in mammals. To study how gene expression is globally regulated by the clock mechanism, we used a high density oligonucleotide probe array (GeneChip) to profile gene expression patterns in Drosophila under light-dark and constant dark conditions. We found 712 genes showing a daily fluctuation in mRNA levels under light-dark conditions, and among these the expression of 115 genes was still cycling in constant darkness, i.e. under free-running conditions. Unexpectedly the expression of a large number of genes cycled exclusively under constant darkness. We found that cycling in most of these genes was lost in the arrhythmic Clock (Clk) mutant under lightdark conditions. Expression of periodically regulated genes is coordinated locally on chromosomes where small clusters of genes are regulated jointly. Our findings reveal that many genes involved in diverse functions are under circadian control and reveal the complexity of circadian gene expression in Drosophila.

No abstract

We searched for immediate early cytokine responsive genes and isolated a novel gene, CIS (Cytokine Inducible SH2 containing protein) that is induced in hematopoietic cells by a subset of cytokines including interleukin-2 (IL-2), IL-3, and erythropoietin (EPO). The mutant IL-2 receptor that fails to activate STAT5 could not induce CIS, suggesting that STAT5 is involved in the cytokine-inducible expression of CIS. We cloned the 5′-flanking region of the CIS gene and found that about 200 bases upstream of the transcription-initiation site contain four potential STAT5 binding sites (MGF boxes). Luciferase reporter assays showed that these MGF boxes were essential for EPO-dependent promoter activity. Expression of STAT5 and the EPO receptor in HEK293 cells conferred EPO-dependent activation of the CIS promoter. These data indicate that CIS is a target of the JAK-STAT5 pathway of cytokine receptors. CIS contains an SH2 domain and binds to tyrosine-phosphorylated EPO and IL-3 receptors. In HEK293 cells expressing STAT5 and the EPO receptor, EPO-dependent tyrosine phosphorylation of STAT5, as well as EPO-dependent CIS-promoter activation, was suppressed when CIS was coexpressed. Moreover, the induction of oncostatin M, another STAT5 target, as well as the tyrosine-phosphorylation of STAT5, were partially suppressed by CIS expression in Ba/F3 cells. Thus, CIS is a feedback modulator of STAT5; its expression is induced by STAT5 and it negatively modulates STAT5 activation.

No abstract