Up-regulation of the urokinase-type plasminogen activator receptor by monocyte chemotactic proteins.

The urokinase-type plasminogen activator receptor (uPAR) is a multifunctional molecule involved in migration and adhesion of leukocytes to sites of inflammation. Based on our hypothesis that a chemoattractant can stimulate uPAR expression by its target cell, thereby promoting cell migration, we employed three chemokines [monocyte chemotactic protein (MCP)-1, MCP-2 and MCP-3] as chemoattractants, and examined their effect on uPAR expression in a human monocyte-like cell line, U937. Northern blot analysis demonstrated that all three chemokines tested increased the level of uPAR mRNA in time-dependent and dose-dependent manners. Among them, MCP-3 exhibited the most potent effect. Scatchard analysis showed that incubation with MCP-3 (1 x 10(-8) mol/l) for 16 h resulted in a significant increase in the number of uPAR from (6.8 +/- 0.3) x 10(3) to (10.3 +/- 1.6) x 10(3)/cell, and in a slight increase in the equilibrium dissociation constant, K(d). The effect of anti-uPAR antibodies on MCP-3-induced U937 cell migration across an endothelial cell monolayer and a type I collagen layer was assessed by means of the modified Boyden chamber assay. Although MCP-3 caused a three-fold increase in migration, incubation with an antibody to uPAR markedly abrogated the induced cell migration. These results support our hypothesis and suggest that up-regulation of uPAR in target cells might be an important and common feature of chemoattractants.

A novel function of extraerythrocytic hemoglobin: identification of globin as a stimulant of plasminogen activator biosynthesis in human fibroblasts.

Following wounding, the surrounding fibroblasts migrate towards the clotted blood in the wounded space to form granulation tissue resulting in wound repair. One of the most abundant proteins in the wound is hemoglobin (Hb). The aim of the present study was to examine the effect of Hb on fibroblasts in producing components of the plasminogen-plasmin system which play an important role in wound healing. Human Hb A0 added to cultures of human fibroblasts elicited a dose-dependent increase in fibrinolytic activity. ELISA demonstrated an increased fibrinolytic activity due to increased urokinase-type plasminogen activator (uPA). An increase in tissue-type PA was also detected, while the type-I PA inhibitor level remained unaffected. Globin showed a similar effect, while hemin and protoporphyrin IX exerted no effect. The influence of Hb was quenched when haptoglobin was added. Although northern blot analysis revealed no difference in uPA transcripts between stimulated and non-stimulated cells, immunoprecipitation experiments confirmed an increased uPA synthesis in Hb- and globin-treated cells, suggesting that enhanced expression is achieved through translational regulation. These findings suggest a potential role for globin in modulating cellular functions during the process of wound healing.

Inhibitory factors in breastmilk, maternal and infant sera against in vitro growth of Plasmodium falciparum malaria parasite.

Complications from falciparum malaria are responsible for over one million infant deaths annually. There is as yet no clinically protective vaccine that has been developed against human malaria parasites. While several studies have demonstrated the inhibitory properties of human sera against Plasmodium falciparum, there is no reported investigation that has examined the protective effects of human breastmilk against the malaria parasite. This study demonstrates the presence of significant antibody titers to ring, trophozoite, schizont and gametocyte stages of P. falciparum in 144 Nigerian maternal milk samples and also in paired maternal and infant sera. The study also demonstrates significant in vitro growth inhibition of P. falciparum by maternal and infant sera, but most notably by breastmilk samples and breastmilk constituents, such as lactoferrin and sIgA. The results therefore suggest a protective in vivo role for breastmilk in the possible modulation of malaria frequency, severity and complications.

Axin, an inhibitor of the Wnt signalling pathway, interacts with beta-catenin, GSK-3beta and APC and reduces the beta-catenin level.

BACKGROUND: The Wnt/Wingless signalling pathway plays an important role in both embryonic development and tumorigenesis. Beta-catenin and Axin are positive and negative effectors of the Wnt signalling pathway, respectively. RESULTS: We found that Axin interacts with beta-catenin and glycogen synthase kinase-3beta (GSK-3beta). Furthermore, the regulation of the G-protein signalling (RGS) domain of Axin is associated with the colorectal tumour suppressor adenomatous polyposis coli (APC). Overexpression of Axin in the human colorectal cancer cell line SW480 induced a drastic reduction in the level of -catenin. Interaction with beta-catenin and GSK-3beta was required for the Axin-mediated beta-catenin reduction. CONCLUSION: Axin interacts with beta-catenin, GSK-3beta and APC, and negatively regulates the Wnt signalling pathway, presumably by regulating the level of beta-catenin.

Inhibition of a snake venom hemorrhagic metalloproteinase by human and rat alpha-macroglobulins.

Jararafibrase I is a hemorrhagic metalloproteinase purified from Bothrops jararaca venom, which induces local hemorrhage by degrading the basement membrane components. The present study was undertaken to investigate the inhibition of jararafibrase I by human and rat serum proteinase inhibitors. The proteolytic activity of jararafibrase I was completely inhibited by human and rat sera. In particular, rat serum displayed a greater inhibitory capacity. The inhibitory capacities of both sera were dependent on alpha-macroglobulins. SDS-PAGE analysis revealed that jararafibrase I formed complexes with alpha-macroglobulins that were present in normal sera. The proteolytic activity of jararafibrase I was completely inhibited by alpha1-macroglobulin and murinoglobulin in rat serum, and by human alpha2-macroglobulin. The inhibition molar ratios of alpha-macroglobulin/jararafibrase I were 1.5 for rat alpha1-macroglobulin and human alpha2-macroglobulin, and 2.4 for rat murinoglobulin. SDS-PAGE under reducing conditions demonstrated that the bait region of human alpha2-macroglobulin and rat murinoglobulin was cleaved by jararafibrase I. The bait region cleavage sites were identified as being situated at the 696Arg-697Leu peptide bond in human alpha2-macroglobulin, and at the 686Ala-687Val peptide bond in rat murinoglobulin.

Activation of single-chain urokinase-type plasminogen activator by a hemorrhagic metalloproteinase, jararafibrase I, in Bothrops jararaca venom.

Urokinase-type plasminogen activator (uPA) activates plasminogen to plasmin, which is involved in the degradation of the vascular basement membrane and extracellular matrix. The present study was undertaken to examine the effects of several hemorrhagic metalloproteinases, jararafibrase (JF) I, II, III and IV, purified from Bothrops jararaca venom, on the single-chain zymogen form of uPA (scuPA). Activation of scuPA by JF I IV was estimated using a synthetic substrate for uPA (S-2444). Only JF I activated the scuPA in a time- and dose-dependent manner. SDS-PAGE analysis revealed that, after incubation with JF I, the intensity of the 55 kDa band of scuPA decreased concomitantly with increases in the intensity of the major two bands at 32 and 22 kDa under reduced and non-reduced conditions. The 32 kDa band demonstrated fibrinolytic activity in fibrin-zymographic studies. Amino-acid-sequence analysis revealed that JF I cleaved the position of 143Glu-144Leu in scuPA, indicating that JF I formed low molecular weight scuPA. From these results, it seems possible that activation of scuPA by JF I could be responsible in part for the local hemorrhage and tissue damage that are frequently observed in human victims of B. jararaca envenoming.

Effects of bromocriptine in Huntington chorea. Case report.

1. The effects of bromocriptine (BC) on choreiform movement were compared with those of bromperidol (BP) and fluphenazine (FLZ) in a patient with Huntington disease. The patient (male, 42 years old) was treated with BP (15 mg/day, 4 weeks), FLZ (3 mg/day, 4 weeks), low dose of BC (5 mg/day, 4 weeks) and relatively high dose of BC (10 mg/day, 8 weeks). The CSF content of homovanilic acid (HVA) was assayed at last day of the each drug trial. The efficacy of the drugs was evaluated by electromyography. 2. Although BP and FLZ did not succeed to ameliorate the choreiform movement, both low dose and high dose BC showed rapid improvement of the involuntary movement. The CSF HVA concentration was 35.0 ng/ml before beginning treatment. Whereas FLZ and high dose of BC substantially increased the levels of HVA after the dosage (49.3 and 53.1 ng/ml, respectively), moderate increase of HVA (41.5 ng/ml) was observed when the low dose of BC was administered. These observations suggest that increase of CSF HVA might be necessary for clinical improvement of choreiform movement but not correlate with the degree of improvement and dopamine agonists could be useful drug for the treatment of choreiform movement which is refractory to the administration of neuroleptics.

Clearance and distribution of a haemorrhagic factor purified from Bothrops jararaca venom in mice.

We previously purified two fibrinolytic/haemorrhagic enzymes (jararafibrase-I and II) from Bothrops jararaca venom. In the present study, the clearance, organ distribution and local absorption rate were examined in mice using 125I-labelled jararafibrase-I. Following intravenous injection of 125I-labelled jararafibrase-I, a complex was rapidly formed with the plasma protein and the radioactivity quickly disappeared from the circulation with a half-life of about 3 min for the initial part of the curve. The highest level of the radioactivity (59.5%) was seen in the liver at 5 min after dosing, and the next highest level of radioactivity (14.4%) was seen in the kidney at 60 min after dosing. At 60 min after dosing, 36.8% of the total injected radioactivity was seen in the contents of the small intestine, and 11.4% of the total injected radioactivity was seen in the contents of the large intestine at 120 min after dosing. It is assumed that the jararafibrase-I was metabolized mainly in the liver, to small mol. wt products, and excreted in the intestine via the bile duct. Also, a small amount of jararafibrase-I appeared to be metabolized in the kidney. Following subcutaneous injection, a high-dose group revealed a low local absorption rate. The low local absorption rate was apparently due to a diminished blood flow caused by subcutaneous haemorrhage.

Early and late results with combined mitral and aortic valve replacement.

From February 1977 to January 1993, a total of 52 patients have undergone combined mitral and aortic valve replacement (MAVR) at the Fukuoka University Hospital. The ages at operation ranged from 35 to 72 years (mean, 55.3 years) for 16 males and 36 females. Twenty-five patients received two bioprosthetic valves (group BB); 6 received a combination of bioprosthetic valve and mechanical valve (group BM); and 21 received two mechanical valves (group MM). The previous intra-cardiac operations were performed on 24 patients (46.2%). The purpose of this study was to learn about the long-term clinical results of MAVR and to analyze the difference among three groups. Seven patients died within 30 days of operation or during initial hospitalization (early mortality: 13.5%). Two patients died at late period, 5.8 years and 6.9 years after operation, respectively (linearized occurrence rate: 0.9%/pt-yr). Patient survival rate including operative death was 82.9% +/- 5.7% at 5 years and 79.0% +/- 6.7% at 8 years in the total number of patients. Thromboembolism occurred in 2 patients in group MM (0.9%/pt-yr). Anticoagulant-related hemorrhage occurred only in 1 patient (0.4%/pt-yr in overall), in whom two bioprosthetic valves were implanted (group BB), in spite of being in good control with anticoagulants. No infective endocarditis was encountered in any patients during any of the periods. Reoperations were performed in 7 patients (3%/pt-yr in overall), 5 in group BB and 2 in group MM. Percentage freedom from reoperation was 86.4% +/- 5.9% at 5 years and 78.2% +/- 7.7% at 8 years in all. There was no difference among the three groups. Percentage freedom from overall morbidity and mortality was 74.5% +/- 6.9% at 5 years in all. Group MM showed higher morbidity and mortality at 8 years than other groups, but there were no significance (MM: 52.4% +/- 17.6%, BB: 76.5% +/- 9.5%, BM: 83.3% +/- 15.2%). We conclude that there were no significant differences in long-term results of NAVR which consisted of the following combinations of prostheses such as BB, BM, and MM. Redoing MAVR with New York Heart Association functional class IV and emergency cases were considered as in-hospital risk factors.

[Fundamental and clinical studies on T-1982 (cefbuperazone) in the field of obstetrics and gynecology].

T-1982 (cefbuperazone), a new cephem antibiotic, was fundamentally and clinically studied in the field of obstetrics and gynecology. The following results were obtained. The concentrations of the drug in arterial and venous blood, and genitalia following intravenous drip infusion were measured. The results demonstrated favourable transfer of the drug into various internal genital organs. Eleven patients with bacterial infections were treated with T-1982. The therapeutic results were markedly effective in 2 and good in 9 cases, therefore the effective rate was 100%. No side effects were noted in any cases. It is therefore, presumed, that T-1982 is a useful drug for infectious diseases in the field of obstetrics and gynecology although the number of subjects was not so large in this study.