Inhibition of plasminogen activator inhibitor-1 by angiotensin II receptor blockers on cyclosporine-treated renal allograft recipients.

INTRODUCTION: We previously showed that proteinuria from a renal graft was significantly decreased by administration of losartan potassium, an angiotensin II receptor blockers (ARB). To further evaluate the mechanism, we performed another clinical study focusing on the change in plasma plasminogen activator inhibitor-1 (PAI-1) levels among cyclosporine (CyA)-treated renal allograft recipients. METHODS: Among 12 hypertensive CyA-treated kidney transplant patients, four received 25 to 50 mg/day of losartan; four, 4 to 8 mg/day of candesartan cilexetil; and another four, 20 to 40 mg/day of nifedipine. Four CyA-treated kidney-transplanted patients without hypertension were selected as a control group. Informed consent was obtained from all participants. PAI-1 and serum creatinine (S-Cr) levels were monitored every 3 months for 1 year. RESULTS: Considering the pretreatment of PAI-1 as 100%, the mean percent of PAI-1 at 1 year after the onset of study for losartan, candesartan, nifedipine, and control groups were 78.6 +/- 6.7%, 81.4 +/- 8.0%, 96.7 +/- 7.6%, and 110.4 +/- 9.2%, respectively. The ARB groups demonstrated significant differences from the control group (P < .01), while the nifedipine group did not. S-Cr levels among ARB-administered groups were increased slightly but temporarily. As for S-Cr levels, no significant differences were seen among the four groups. CONCLUSIONS: Control of hypertension itself is important for all renal graft recipients; however, PAI-1 reduction by ARBs was thought to be a key for renal preservation. We expect that ARBs will contribute to prolonged renal allograft survival.

Survival of memory T cells specific for Japanese cypress pollen allergen is maintained by cross-stimulation of putative pectate lyases from other plants.

In view of recent studies on the mechanisms of the survival of peripheral memory T cells, we tested the biologic role of pectate lyase, a pectin-degrading enzyme, as the cross-reactive antigen required for the recurring survival signals for human T cells specific for Cha o 1, a pollen allergen molecule of the Japanese cypress. We determined a 16-mer epitope peptide for the T-cell clone, and prepared synthetic oligopeptides of homologous regions in putative pectate lyase of other plants. Of these homologous peptides, ZePel (Zinnia elegans), ban 17 (banana), and Amb a 1.1 (short ragweed) induced strong proliferative responses of the Cha o 1-specific T-cell clone in vitro. In addition, suboptimal doses of peptide homologs derived from banana and short ragweed enhanced the survival potency of this T-cell clone without detectable proliferative responses to the peptides. When there was no antigen stimulation, the T-cell clone decreased in viable cell number and lost antigen-specific proliferation activity on day 6 during in vitro incubation. On the other hand, T-cell clones incubated with these survival-inducing peptides maintained proliferative activity to Cha o 1 even on day 9. Serum derived from the donor patient did not contain detectable levels of IgE specific to banana or short ragweed by CAP-RAST. These results show that human T cells specific for pollen allergen seem to use cross-reactive pectate lyase peptides to deliver survival signals even in the absence of pollen allergen, and memory T cells maintained in such a manner might be functioning at the onset of allergic pollinosis, although pollen allergens are seasonal.

The first total synthesis of (+/-)-linderol A, a tricyclic hexahydrodibenzofuran constituent of Lindera umbellata bark, with potent inhibitory activity on melanin biosynthesis of cultured B-16 melanoma cells.

[reaction in text] The first total synthesis of (+/-)-linderol A, a hexahydrodibenzofuran isolated from Lindera umbellata bark, with potent inhibitory activity on melanin biosynthesis of cultured B-16 melanoma cells was achieved via a 20-step of reaction in 7.64% overall yield starting from 4,6-dimethoxysalicylaldehyde.

The role of aldose reductase in sugar cataract formation: aldose reductase plays a key role in lens epithelial cell death (apoptosis).

Since aldose reductase is localized primarily in lens epithelial cells, osmotic insults induced by the accumulation of sugar alcohols occur first in these cells. To determine whether the accumulation of sugar alcohols can induce lens epithelial cell death, galactose-induced apoptosis has been investigated in dog lens epithelial cells. Dog lens epithelial cells were cultured in Dulbecco's modified Eagle's mimimum essential medium (DMEM) supplemented with 20% fetal calf serum (FCS). After reaching confluence at fifth passage, the medium was replaced with the same DMEM medium containing 50 mM D-galactose and the cells were cultured for an additional 2 weeks. Almost all of the cells cultured in galactose medium were stained positively for apoptosis with the terminal deoxynucleotidyl transferance-mediated biotin-dUTP nick end labeling (TUNEL) technique. Agarose gel electrophoresis of these cells displayed obvious DNA fragmentation, known as a ladder formation. All of these apoptotic changes were absent in similar cells cultured in galactose medium containing 1 microM of the aldose reductase inhibitor AL 1576. Addition of AL 1576 also reduced the cellular galactitol levels from 123+/-10 microgram/10(6) cells (n=5) to 3.9+/-1.9 microgram/10(6) cells (n=5). These observations confirm that galactose induced apoptosis occurs in dog lens epithelial cells. Furthermore, the prevention of apoptosis by an aldose reductase inhibitor suggests that this apoptosis is linked to the accumulation of sugar alcohols.

Vaccination with calpain induces a Th1-biased protective immune response against Schistosoma japonicum.

A large subunit of calpain, a calcium-activated neutral proteinase, from Schistosoma japonicum was cloned and expressed in Escherichia coli. When BALB/c mice were immunized with purified recombinant calpain (r-calpain) emulsified in complete Freund's adjuvant, a significant reduction in the number of recovered worms and also in egg production per female worm was observed (P<0.01). Spleen cells of the immunized mice showed enhanced production of gamma interferon (IFN-gamma) by activated CD4(+) T cells. Considering our observation of elevated expression of inducible nitric oxide synthase mRNA in immunized mice, r-calpain-induced IFN-gamma seemed to upregulate the production of nitric oxide by macrophages and subsequently mediated the killing of schistosomulae in the lung. On the other hand, spleen cells of immunized mice showed only faint interleukin-4 production in response to r-calpain in vitro, suggesting that immunization with r-calpain alters the Th1-Th2 balance in murine hosts even during a Th2-promoting S. japonicum infection. Furthermore, histopathological study of the livers of immunized mice showed that granulomas formed around eggs were diminished in both size and number. Egg production by female worms was clearly decreased in immunized mice, suggesting that r-calpain also has antifecundity effects. Taken together, these results point to S. japonicum calpain as a potential vaccine candidate for both worm killing and disease prevention, possibly through the induction of a strong Th1-dominant environment in immunized mice.

Trypanocidal effects of gallic acid and related compounds.

Gallic acid (3,4,5-trihydroxybenzoic acid) is a naturally abundant plant phenolic compound and it is well known as a component of hydrolyzable tannins. We report here that gallic acid and related compounds have trypanocidal activity against Trypanosoma brucei brucei (GUTat 3.1) in both the long slender bloodstream forms and the procyclic forms, in vitro. LD50 values of gallic acid are 46.96 +/- 1.28 microM for bloodstream forms and 30.02 +/- 3.49 for procyclic forms, respectively. A study of structurally related compounds suggested that the pyrogallol moiety could be responsible for this activity.

Antitrypanosomal effects of traditional Chinese herbal medicines on bloodstream forms of Trypanosoma brucei rhodesiense in vitro.

The antitrypanosomal activity of traditional Chinese herbal medicines and these crude drug ingredients were determined using axenic cultured bloodstream forms of Trypanosoma b. rhodesiense which is one of the two causative agents of African sleeping sickness in man. The drugs tested were 8 traditional Chinese herbal medicines and these 14 crude drug ingredients. Of these traditional Chinese medicines examined, san'o-shasin-to and oren-gedoku-to showed most potent antitrypanosomal effect. The minimal effective concentration (MEC) which killed all bloodstream form populations within 24 hours of both drug exposure was 125 microg/ml. The 50% effective concentration (EC50) of san'o-shashin-to and oren-gedoku-to was 63 and 74 microg/ml, respectively. In the crude drug ingredients tested, Scutellaria baicalensis G. and Coptis japonica M. which are the main components of san'o-shasin-to and oren-gedoku-to, showed the most powerful antitrypanosomal activity. The MEC and EC50 value of these crude drug ingredients were 30 and 60 microg/ml, and 20 and 36 microg/ml.

Mutated mitogen-activated protein kinase: a tumor rejection antigen of mouse sarcoma.

The molecular basis of the polymorphic tumor rejection antigens of chemically induced sarcomas of inbred mice remains a mystery, despite the discovery of these antigens over 40 years ago and their critical importance to the foundation of tumor immunology. In an analysis of a panel of BALB/c 3-methylcholanthrene-induced tumors, we identified one tumor, CMS5, that elicited a strong cytotoxic T cell response with exquisite specificity for CMS5. A stable cloned line of T cells with this specificity (C18) was used to screen a CMS5 cDNA expression library. The gene encoding the C18-defined antigen was identified as a mutated form of a mouse mitogen-activated protein kinase, ERK2, and a peptide incorporating the resulting amino acid substitution (lysine to glutamine) was efficiently recognized by C18. Vaccination with this peptide elicited specific resistance to CMS5 challenge. Extensive efforts to isolate antigen-loss variants of CMS5 were unsuccessful, suggesting that the mutated mitogen-activated protein kinase is essential for maintenance of the malignant phenotype.

Purification, characterization and molecular cloning of Cha o 1, a major allergen of Chamaecyparis obtusa (Japanese cypress) pollen.

Pollen of Chamaecyparis obtusa (Japanese cypress) is one of the causes of allergic pollinosis in Japan. A major allergen of the pollen designated Cha o 1, was purified by two-step ion exchange chromatography. Cha o 1 was separated into four components with molecular masses of 48.5 kDa and 52.0 kDa, each with pIs of 6.77 and 6.82. The 23-residue N-terminal sequence of Cha o 1 was determined and shown to have high identity with that of Cry j 1, a major allergen of Cryptomeria japonica pollen. cDNA coding for Cha o 1 was cloned by hybridization screening using Cry j 1 cDNA as a probe. One of the cDNA clones, pCHA-1 was sequenced and found to code for a putative 21-residue signal peptide and a 354-residue native protein with a derived molecular mass of 38.1 kDa. The deduced amino acid sequence of Cha o 1 showed 79-80% identity with those of Cry j 1. These findings were consistent with observations of a close crossreaction between the two allergens. Homology analyses revealed that Cha o 1 had 46-49% identity with Amb a 1 families and Amb a 2, the major allergens of short ragweed. Cry j 1 has pectate lyase enzyme activity, suggesting that Cha o 1 may have the same enzyme activity as Cry j 1.

Antigenic analysis of Cryptomeria japonica and Chamaecyparis obtusa using anti-Cry j 1 monoclonal antibodies.

In Japan, pollen of Cryptomeria japonica and Chamaecyparis obtusa are a yearly source of distress for many people suffering seasonally from allergic rhinitis. To study common epitopes shared by the two species, two monoclonal antibodies (moAbs) were raised against Cry j 1, which is the most predominant allergen in C. japonica. One of the moAbs was found to be reactive even to the major allergen of C. obtusa, demonstrating that the moAb (J1B01) can detect an epitope shared by both species J1B01 strongly inhibited the binding of the major allergens of C. japonica and C. obtusa to IgE of patients who are sensitive to C. japonica and C. obtusa. This finding signifies the importance of the epitope recognized by J1B01.

[Clinical characterization of allergic patients sensitized to Chamaecyparis obtusa--using AlaSTAT system].

Clinical characterization of patients with allergy to Chamaecyparis obtusa (Cypress) pollen and their specific IgE antibody was investigated by using AlaSTAT assay, a new method for measurement of antigen-specific IgE antibody. Positive rate of antigen-specific antibody in patients with allergic pollinosis was 83.5% for Japanese cedar pollen and 80.0% for Chamaecyparis obtusa pollen. 76.4% of patients were sensitized both to Japanese cedar and Chamaecyparis obtusa pollen, however, 7.9% were sensitized only to the latter. As for clinical symptoms, duration of attack was short and severity was mild or moderate in the patients sensitized only to Chamaecyparis obtusa pollen. Patients who visited hospital in April, in that Chamaecyparis obtusa pollen disseminated in the air predominantly, showed significant elevation in AlaSTAT scores compared with patients who visited in February and March (p < 0.05).

The mechanism of photosensitization in photodynamic therapy: phosphorescence behavior of porphyrin derivatives in saline solution containing human serum albumin.

The phosphorescence properties, especially the dynamic behavior of metal free and metal complexed porphyrins, have been studied in phosphate buffered saline (PBS) containing 0-3% human serum albumin (HSA). 6,7-Bisaspartyl-2,4-bis (1-hexyloxyethyl)-deutero- porphyrin (DP) and its gallium(III), zinc(II), and indium(III) complexes are used as photosensitizers. Upon irradiation, a solution of porphyrins containing more than 0.1% HSA shows phosphorescence with a lifetime longer than 1 ms. With an increase in irradiation time, phosphorescence intensities and lifetimes of porphyrins increase, depending upon their concentrations and triplet lifetimes, and approach saturated values close to those under deaerated conditions. The experimental results may be interpreted in terms of hypoxia induced by photosensitization in a local environment surrounding the sensitizer. The hypoxia is caused by the reaction between proteins and singlet molecular oxygen generated by photosensitization of porphyrins. Phosphorescence behavior of sensitizers in HSA PBS solution gives significant information for classifying photosensitizers as to their efficacy for photodynamic therapy.