ABSTRACT Thermococcus kodakarensis possesses two chaperonins, CpkA and CpkB, and their expression is induced by the downshift and upshift, respectively, of the cell cultivation temperature. The expression levels of the chaperonins were examined by using specific antibodies at various cell growth temperatures in the logarithmic and stationary phases. At 60°C, CpkA was highly expressed in both the logarithmic and stationary phases; however, CpkB was not expressed in either phase. At 85°C, CpkA and CpkB were expressed in both phases; however, the CpkA level was decreased in the stationary phase. At 93°C, CpkA was expressed only in the logarithmic phase and not in the stationary phase. In contrast, CpkB was highly expressed in both phases. The results of reverse transcription-PCR experiments showed the same growth phase- and temperature-dependent profiles as observed in immunoblot analyses, indicating that the expression of cpkA and cpkB is regulated at the mRNA level. The cpkA or cpkB gene disruptant was then constructed, and its growth profile was monitored. The cpkA disruptant showed poor cell growth at 60°C but no significant defects at 85°C and 93°C. On the other hand, cpkB disruption led to growth defects at 93°C but no significant defects at 60°C and 85°C. These data indicate that CpkA and CpkB are necessary for cell growth at lower and higher temperatures, respectively. The logarithmic-phase-dependent expression of CpkA at 93°C suggested that CpkA participates in initial cell growth in addition to lower-temperature adaptation. Promoter mapping and quantitative analyses using the Phr ( Pyrococcus heat-shock regulator) gene disruptant revealed that temperature-dependent expression was achieved in a Phr-independent manner.
Polyclonal and monoclonal anti-tissue plasminogen activator (t-PA) antibodies were characterized by using enzyme immunoassay (EIA) in which β-D-galactosidase was coupled to anti-t-PA antibody (Fab'). 2:2 B10 and 1:3 G5 antibodies, specific for both one-chain and two-chain t-PA, strongly bound with one-chain t-PA purified from cultured melanoma cell lines, but 1:3 C5 antibody bound weakly with such t-PA. When polyclonal t-PA antibody was used as the first reaction antibody immobilized on silicone pieces, anti-t-PA polyclonal antibody mainly reacted with 2:2 B10 or 1:3 C5 antigenic determinant. When t-PA levels in the plasma were determined, the presence of EDTA enhanced the sensitivity of t-PA determination by the present EIA technic. 2:2 B10 monoclonal antibody detected a part of t-PA molecules in the plasma that polyclonal antibody detected. T-PA was mainly detected in the endothelial cells, but not in the muscular layer of inferior mesenteric artery when immunochemical technic was used where polyclonal t-PA antibody was applied.
Release of Bβ peptides and F (g) DP from fibrin (ogen) was studied after the activation of Glu-plasminogen (Glu-plg) by urokinase (UK) in the plasma or clot and fibrinogen or fibrin. Bβ peptides or FDP were released faster from the clot than the plasma. In a purified system, FDP was released faster than FgDP after the activation of Glu-plg by UK in the presence of fibrin or fibrinogen. Release of Bβ 15-42 from purified fibrin was slower than the release of Bβ 1-42 from fibrinogen when Glu-plg was activated by UK. The presence of α2 antiplasmin (α2AP) slowed the release of Bβ 1-42 from fibrinogen, thus resulted in faster release of Bβ 15-42 in comparison to Bβ 1-42. These results indicated that Glu-plg was activated better by UK in the presence of fibrin than fibrinogen, but the release of Bβ peptides from purified fibrin and fibrinogen depended upon the presence of α2AP. Since fibrin prevented inactivation of plasmin by α2AP, the presence of α2AP inactivated plasmin in the fluid phase.
Plasma concentration of fibrinogen (Fbg), plasminogen (PLG), antithrombin III (AT III), alpha 2-plasmin inhibitor (alpha 2-PI), thrombin antithrombin III complex (TAT) and plasmin alpha 2-plasmin inhibitor complex (PIC) were evaluated in 23 nephrotic patients with proteinuria more than 3.5 g/day, including 4 cases with clinical evidence of thromboembolism. Among patients without thromboembolism, concentration of PLG and AT III was in the normal range but that of Fbg and alpha 2-PI was significantly elevated (p less than 0.01 for Fbg and p less than 0.001 for alpha 2-PI respectively). Also there was a positive relationship between AT III and serum albumin (p less than 0.05). Two fifth of these patients had an had an increased level of TAT, and also had a higher level of PIC compared with normal control (p less than 0.01). There was a positive relationship between TAT and PIC, TAT and Fbg (p less than 0.05), PIC and Fbg (p less than 0.01). TAT and PIC levels were markedly elevated in the patients with thromboembolism. From aforementioned data, it was suggested that patients with nephrotic syndrome would be in the prethrombotic state and the increased level of Fbg is one of the major risk factors of thromboembolic complications in these patients. Furthermore measurement of TAT and PIC are the useful means for the diagnosis of these complications.
To investigate if neurotensin (NT) could induce activation of urokinase‐type plasminogen activator (uPA) in vascular endothelial cells, we utilized the acetyl‐NT (8–13) analogue, TJN‐950, in which the C‐terminal leucine is reduced to leucinol. TJN‐950 inhibited the binding of 125 I‐NT to membranes of newborn rat brains and of COS‐7 cells transfected with rat NT receptor cDNA, but at 10 4 higher doses than NT (8–13). However, TJN‐950 was as effective as NT in inducing the fibrinolytic activity in bovine vascular aortic and human umbilical vein endothelial cells, and enhanced the migration of vascular endothelial cells. Moreover, administration of TJN‐950 induced neovascularization in the rat cornea in vivo. TJN‐950 had no effect on expression of uPA, plasminogen activator inhibitor‐1 or uPA receptor mRNA. The binding of 125 I‐TJN‐950 to cell membranes was blocked by unlabeled uPA and TJN‐950, but not the amino‐terminal or 12–32 fragment of uPA. TJN‐950 may enhance uPA activity in vascular endothelial cells by interacting with the uPA receptor, resulting in induction of angiogenesis.
