Signal transducer and activator of transcription (STAT)-induced STAT inhibitor 1 (SSI-1)/suppressor of cytokine signaling 1 (SOCS1) inhibits insulin signal transduction pathway through modulating insulin receptor substrate 1 (IRS-1) phosphorylation.

Signal transducer and activator of transcription (STAT)-induced STAT inhibitor 1 (SSI-1) is known to function as a negative feedback regulator of cytokine signaling, but it is unclear whether it is involved in other biological events. Here, we show that SSI-1 participates and plays an important role in the insulin signal transduction pathway. SSI-1-deficient mice showed a significantly low level of blood sugar. While the forced expression of SSI-1 reduced the phosphorylation level of insulin receptor substrate 1 (IRS-1), SSI-1 deficiency resulted in sustained phosphorylation of IRS-1 in response to insulin.Furthermore, SSI-1 achieves this inhibition both by binding directly to IRS-1 and by suppressing Janus kinases. These findings suggest that SSI-1 acts as a negative feedback factor also in the insulin signal transduction pathway through the suppression of IRS-1 phosphorylation.

Context-dependent modulation of replication activity of Saccharomyces cerevisiae autonomously replicating sequences by transcription factors.

Evidence for transcription factor involvement in the initiation of DNA replication at certain replication origins in Saccharomyces cerevisiae mainly comes from an indirect assay which measures the mitotic stability of plasmids containing an autonomously replicating sequence (ARS), a selectable marker gene, and a centromere. In order to eliminate the effect of transcription factor binding to the selectable marker gene or centromere in such assays, we have adapted the DpnI assay to directly measure ARS replication activity in vivo by using ARS plasmids devoid of extraneous transcription elements. Using this assay, we found that the B3 element of ARS1, which serves as a binding site for the transcription factor Abf1p, does not stimulate ARS activity on plasmids lacking a centromere and a selectable marker gene. We also found with such plasmids that exogenous expression of the strong transcriptional activators Gal4 and Gal4-VP16 inhibited the replication activity of ARS1 when B3 was replaced by the Gal4 binding site, although these activators had previously been shown to stimulate replication activity in the stability assay. Moreover, a chromosomally inactive ARS, ARS301, which was active by itself on a plasmid, was inactivated by placing an Abf1p binding site in its vicinity. These results indicate that the sequences surrounding the ARS as well as properties of the ARS element itself determine its response to transcription factors.

Block of granulocytic differentiation of 32Dcl3 cells by AML1/ETO(MTG8) but not by highly expressed Bcl-2.

The chimeric gene, AML1/ETO (MTG8), generated in t(8;21) acute myeloid leukemia enhances the expression of Bcl-2. To evaluate whether this enhancement is the primary role of AML1/ETO in leukemogenesis, effects of over-expression of Bcl-2 in the murine myeloid precursor cell line, 32Dcl3, were examined. When 32Dcl3 cells expressing exogenous Bcl-2 were induced to differentiate, the onset of morphological differentiation was delayed. However, even the cells expressing very high levels of exogenous Bcl-2 eventually underwent differentiation without a significant decrease in the synthesis of Bcl-2. On the contrary, 32Dcl3 cells stably expressing AML1/ETO were completely resistant to differentiation and continued to grow in the presence of G-CSF. These results are consistent with the interpretation that stimulation of Bcl-2 expression is not the primary target of AML1/ETO.

Purification and characterization of four Ca(2+)-dependent lectins from the marine invertebrate, Cucumaria echinata.

Four Ca(2+)-dependent, N-acetylgalactosamine/galactose-specific lectins were purified from the marine invertebrate, Cucumaria echinata (Holothuroidea), by column chromatography on lactosyl-Sepharose 4B, Sephacryl S-200, and Q-Sepharose. The molecular masses of these lectins were estimated to be 27 kDa (CEL-I), 35 kDa (CEL-II), 45 kDa (CEL-III), and 68 kDa (CEL-IV) on SDS-PAGE under nonreducing conditions. Among these lectins, CEL-I and CEL-IV strongly agglutinated rabbit and human erythrocytes, and were found to recognize N-acetylgalactosamine and galactose-containing carbohydrates from the results of a hemagglutination inhibition assay. In contrast, CEL-II failed to agglutinate any erythrocytes tested, although its carbohydrate-binding ability was confirmed by a carbohydrate-binding assay involving asialofetuin-horseradish peroxidase. Interestingly, CEL-III caused hemolysis of rabbit and human erythrocytes, while it showed only hemagglutination of chicken and horse erythrocytes at relatively high concentrations. The hemolytic activity of CEL-III was also dependent on the Ca(2+)-concentration, and inhibited by N-acetylgalactosamine and galactose-containing carbohydrates, suggesting that the hemolysis was caused by Ca(2+)-dependent binding of CEL-III to specific carbohydrate chains on the erythrocyte surface and the following partial destruction of the membrane.

A microassay for proteases using succinylcasein as a substrate.

A photometric assay for proteases has been developed. A chemically modified casein whose amino groups were succinylated was used as a substrate. After incubation with trypsin, chymotrypsin, thermolysin, and subtilisin, the extent of hydrolysis of the substrate was determined with trinitrobenzene sulfonate (TNBS). The whole procedure of the assay was performed in the microtiter plate wells and the increase in the absorbance resulting from the reaction between TNBS and newly formed amino groups in the substrate was able to be determined with a high sensitivity by a microtiter plate reader, enabling the simultaneous measurement of a number of samples. Application of this method to the measurement of proteolytic activity contained in the protein extract of Tapes philippinarum is demonstrated.

Effects of cetiedil on the oxidative metabolism of activated polymorphonuclear leucocytes.

Cetiedil, alpha-cyclohexyl-3-thiopheneacetic acid 2-(hexahydro-lH-azepin-l-yl)-ethyl ester, was found to specifically suppress oxygen uptake by polymorphonuclear leucocytes (PMN) that were exposed to myristate or heat-killed E. coli. The chemical had no effect on the basal respiration rate of PMN in the resting state. Inhibition of oxygen uptake by cetiedil was proportionate to the degree of inhibition of the generation of O-2 and H2O2. It was also found that cetiedil suppressed the rate of the phagocytosis by PMN of opsonized oil droplets. Cetiedil had no effect on subcellular NADPH oxidase, an enzyme responsible for the respiratory burst that is activated by the perturbation of PMN plasma membrane with phagocytable particles or stimulators such as myristate. These results suggest that cetiedil affects the trigger mechanism of the plasma membrane to inhibit the activation of NADPH oxidase.

c-Jun stimulates origin-dependent DNA unwinding by polyomavirus large Tantigen.

The AP1 protein c-Jun has previously been shown to stimulate polyomavirus (Py) DNA replication in vivo. In order to define the mechanism, we added purified c-Jun protein to the origin-dependent and large T antigen (LT)-dependent in vitro DNA unwinding assay. c-Jun protein was found to stimulate by approximately 5-fold the unwinding of a 290 bp linear DNA fragment containing both the Py origin and the AP1 recognition sequence to which c-Jun binds. Efficient levels of stimulation were specifically observed at limiting concentrations of LT for unwinding. Under similar conditions, Py DNA replication was stimulated to a comparable extent by AP1 in a purified in vitro replication assay. Mobility shift and DNase I footprinting assays showed that c-Jun stimulates the ATP-dependent binding of LT to the origin core by approximately 7-fold. Furthermore, c-Jun was found to interact directly with LT, but not with replication protein A. The activities of c-Jun to stimulate unwinding and origin binding of LT were found to be harbored within the N-terminal region of c-Jun, which is distinct from the DNA binding domain. We speculate that certain transcription factors may possess specific DNA replication domains that function to stimulate the loading of replication factors at the origin during the initiation of DNA synthesis.

SOCS-1/SSI-1-deficient NKT cells participate in severe hepatitis through dysregulated cross-talk inhibition of IFN-gamma and IL-4 signaling in vivo.

Suppressor of cytokine signaling-1 (SOCS-1), also known as STAT-induced STAT inhibitor-1 (SSI-1), is a negative feedback molecule for cytokine signaling, and its in vivo deletion induces fulminant hepatitis. However, elimination of the STAT1 or STAT6 gene or deletion of NKT cells substantially prevented severe hepatitis in SOCS-1-deficient mice, while administration of IFN-gamma and IL-4 accelerated its development. SOCS-1 deficiency not only sustained IFN-gamma/IL-4 signaling but also eliminated the cross-inhibitory action of IFN-gamma on IL-4 signaling. These results suggest that SOCS-1 deficiency-induced persistent activation of STAT1 and STAT6, which would be inhibited by SOCS-1 under normal conditions, may induce abnormal activation of NKT cells, thus leading to lethal pathological changes in SOCS-1-deficient mice.