Early signs of lung fibrosis after in vitro treatment of rat lung slices with CdCl2 and TGF-beta1.

Precision-cut rat lung slices have been employed in combination with an extensive immunohistochemistry of paraffin-embedded slices for monitoring of early pathohistological changes after exposure to CdCl(2)/TGF-beta(1). Three days of CdCl(2) exposure in combination with TGF-beta(1) seem to be sufficient to induce lung injury with alterations similar to changes observed in early lung fibrogenesis: (1) extracellular matrix accumulation and myofibroblast transdifferentiation (Sirius red staining, collagen type IV, alpha-smooth muscle actin), (2) type I cell injury with loss of type I cell antigens (T1alpha antigen, aquaporin-5, RAGE), (3) increased apoptosis of pulmonary cells (active caspase-3, vimentin cleavage product V1 of caspase-9), and (4) activation of microvascular endothelial cells (podocalyxin, caveolin-1). Western blot analysis confirmed the increasing amount of alpha-smooth muscle actin, the loss of T1alpha antigen, and the increase in caveolin-1 immunoreactivity. The explant culture using CdCl(2)/TGF-beta(1) provides a suitable tool for the study of other factors involved in pulmonary pathology including transcription factors, cytokines, and other metabolites involved in early stages of fibrogenesis.

Suppression of CYP3A2 mRNA expression in the warfarin-resistant roof rat, Rattus rattus: possible involvement of cytochrome P450 in the warfarin resistance mechanism.

1. The continual use of warfarin as a rodenticide has caused the development of populations of warfarin-resistant roof rat. To study the biochemical mechanism of warfarin resistance, the mRNA expression levels of the major P450 forms in the warfarin-resistant and -susceptible roof rat liver following exposure to warfarin were quantified by competitive RT-PCR. 2. The constitutive levels of CYP2C11 and CYP3A2 mRNAs in the warfarin-resistant and -susceptible roof rat were extremely low compared with those in the STD rat. In response to warfarin administration, the CYP3A2 mRNA level in the warfarin-susceptible rat increased to about 3-fold of that before the treatment, whereas in the warfarin-resistant roof rat, CYP3A2 mRNA remained at a low level. 3. The present results suggest the possibility that reduced synthesis of CYP3A2 mRNA is involved in the warfarin-resistant mechanism in the roof rat.

Identification of a caspase-9 substrate and detection of its cleavage in programmed cell death during mouse development.

The caspase family of proteases represents the main machinery by which apoptosis occurs. In vitro studies have revealed that upstream caspases are activated in response to apoptotic stimuli, and the active caspases in turn process downstream effector caspases that are involved in the destruction of cellular structure. Caspase-9 is an upstream caspase that can become active in response to cellular damage, including deprivation of growth factors and exposure to oxidative stress in vitro. Little is known, however, about how activation of caspase-9 is temporally and spatially regulated in vivo, e.g. during development. We have identified vimentin as the first example of a caspase-9 substrate that is not a downstream procaspase. Immunohistochemical analysis, using a specific antibody against the vimentin fragments generated by caspase-9, showed that caspase-9 cleaves vimentin in apoptotic cells in the embryonic nervous system and the interdigital regions. This result is consistent with observations that gene knockouts of caspase-9 and its activator, Apaf-1, result in developmental defects in these tissues. Our results show that the specific antibody is useful for in situ detection of caspase-9 activation in programmed cell death.

Caspase-resistant vimentin suppresses apoptosis after photodynamic treatment with a silicon phthalocyanine in Jurkat cells.

Oxidative stress, such as photodynamic therapy, is an apoptosis inducer. Apoptosis, as well as photosensitization, have been associated with disruption of the cytoskeletal network. The purpose of the present study was to assess the role of vimentin, a major cytoskeletal protein, in apoptosis after photodynamic treatment (PDT) with the silicon phthalocyanine Pc 4 in human Jurkat T cells. Here we show for the first time that photosensitization with Pc 4 initiates vimentin cleavage and that this event precedes poly(ADP-ribose) polymerase (PARP) degradation. Similar findings were obtained in the presence of C2-ceramide, an inducer of oxidative stress and apoptosis. In the presence of benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fluoromethylketone, a pan-caspase inhibitor, Pc 4-PDT-induced vimentin and PARP cleavage were abolished. In Jurkat cells transfected with a caspase-resistant vimentin apoptosis was partly suppressed and delayed post-Pc 4-PDT. We suggest that the full-length vimentin confers resistance to nuclear apoptosis after PDT with Pc 4.

Biochemical characterization of bovine lactoferrin as a glycyrrhizin-binding protein in vitro.

Lactoferrin (LF) from bovine colostrum was biochemically characterized as a glycyrrhizin (GL)-binding protein (gbP) in vitro. It was found that (i) bovine LF (bLF) and a synthetic bovine lactoferricin (bLFcin, the N'-terminal region of bLF at the positions 17--41) had a high affinity to a GL-affinity column; (ii) approximately 1.8 moles of GL were bound to a molecule of bLF with a binding constant of approx. 1.20x10(4) M(-1) at pH 6.8; and (iii) GL, but not glycyrrhetinic acid (GA), induced a conformational change of bLF. In addition, the glucuronic acid moiety of the GL molecule was found to be responsible for binding to bLF, because (i) no binding of GA and two glucoses-GA (Glc-Glc-GA) to bLF was detected; and (ii) a synthetic fluorinated GL (GlcA-GlcF-GA) and mono-glucuronyl-GA (mono-GlcA-GA) were bound significantly to bLF. A similar binding of GL to human LF (hLF) was also observed under the same experimental conditions. Data provided here suggest that (i) bLF contains plural GL-binding sites; and (ii) the specific binding of GL to bLF may modulate the physiological activity of bLF in vivo.

Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-beta.

Apoptosis, or cellular suicide, is important for normal development and tissue homeostasis, but too much or too little apoptosis can also cause disease. The family of cysteine proteases, the so- called caspases, are critical mediators of programmed cell death, and thus far 14 family members have been identified. Some of these, such as caspase-8, mediate signal transduction downstream of death receptors located on the plasma membrane. Others, such as caspase-9, mediate apoptotic signals after mitochondrial damage. Stress in the endoplasmic reticulum (ER) can also result in apoptosis. Here we show that caspase-12 is localized to the ER and activated by ER stress, including disruption of ER calcium homeostasis and accumulation of excess proteins in ER, but not by membrane- or mitochondrial-targeted apoptotic signals. Mice that are deficient in caspase-12 are resistant to ER stress-induced apoptosis, but their cells undergo apoptosis in response to other death stimuli. Furthermore, we show that caspase-12-deficient cortical neurons are defective in apoptosis induced by amyloid-beta protein but not by staurosporine or trophic factor deprivation. Thus, caspase-12 mediates an ER-specific apoptosis pathway and may contribute to amyloid-beta neurotoxicity.

Changes in nuclear morphology during apoptosis correlate with vimentin cleavage by different caspases located either upstream or downstream of Bcl-2 action.

BACKGROUND: Upon Fas stimulation, procaspase-8 is recruited to the death-inducing signalling complex where autoactivation of caspase-8 occurs. Active caspase-8 can directly activate downstream caspases (e.g. caspase-3, 6, and 7) for the execution of apoptosis (mitochondria-independent pathway), while caspase-8 can also lead to executioner caspase activation through mitochondrial damage (mitochondria-dependent pathway). Caspase activation results in the dismantling of intracellular structure through specific proteolysis. RESULTS: We have found that an intermediate filament protein, vimentin, is cleaved at multiple sites by caspases at an early stage of apoptosis in Jurkat cells. The sequences of the two major cleavage sites in vimentin (IDVD/V and DSVD/F) suggested that these sites are cleaved by caspase-8 and caspase-3, respectively, or by close homologues of these proteases. The IDVD/V site can be cleaved by caspase-8 in vitro, and its cleavage is less sensitive to DEVD-CHO and Bcl-2 over-expression than that of the DSVD/F site in Jurkat cells. Over-expression of a mutant vimentin which was insensitive to caspase cleavage at these sites delayed the appearance of apoptotic nuclei in Jurkat cells. CONCLUSION: The specific cleavage of vimentin can be used as an apoptotic marker of both apical- and mitochondria-dependent caspase activation. Apoptotic cleavage of vimentin most likely results in disruption of its filamentous structure, which may facilitate nuclear condensation and subsequent fragmentation through disruption of the cytoskeletal network.

Stable association of 70-kDa heat shock protein induces latent multisite specificity of a unisite-specific endonuclease in yeast mitochondria.

The multisite-specific endonuclease Endo.SceI of yeast mitochondria is unique among endonucleases because its 50-kDa subunit forms a stable dimer with the mitochondrial 70-kDa heat shock protein (mtHSP70), which otherwise fulfills a chaperone function by binding transiently to unfolded proteins. Here we show that the mtHSP70 subunit confers broader sequence specificity, greater stability, and higher activity on the 50-kDa subunit. The 50-kDa subunit alone displayed weaker activity and highly sequence-specific endonuclease activity. The 50-kDa protein exists as a heterodimer with mtHSP70 in vivo, allowing Endo.SceI to cleave specifically at multiple sites on mitochondrial DNA. Endo.SceI may have evolved from a highly specific endonuclease that gained broader sequence specificity after becoming a stable partner of mtHSP70.

Two distinct mechanisms operate in the reactivation of heat-denatured proteins by the mitochondrial Hsp70/Mdj1p/Yge1p chaperone system.

The yeast mitochondrial Hsp70, Ssc1p, functions as a molecular chaperone with its partner proteins, Mdj1p (DnaJ homologue) and Yge1p (GrpE homologue). We have purified a mature form of Ssc1p from yeast mitochondria and those of Mdj1p and Yge1p from Escherichia coli overexpresser cells. With these purified components of the mitochondrial Hsp70 chaperone system, we have succeeded in reconstituting their chaperone functions in the protection of firefly luciferase against thermal damage in vitro. Heat-denatured luciferase is prevented from irreversible aggregation and is maintained in a refolding-competent state by Ssc1p and/or Mdj1p at 42 degreesC. Luciferase denatured at 42 degreesC is actively reactivated by Ssc1p, Mdj1p and/or Yge1p after lowering the temperature to 25 degreesC. The reactivation process of heat-denatured luciferase shows two-phase kinetics. The slow refolding process requires either Ssc1p or Mdj1p at 42 degreesC but the presence of Ssc1p, Mdj1p and Yge1p, and ATP hydrolysis, is essential at 25 degreesC. The slow refolding of luciferase involves multiple rounds of formation and dissociation of the complex between luciferase and Mdj1p/Ssc1p. On the other hand, the fast refolding process is most enhanced when luciferase is incubated with Ssc1p alone at 42 degreesC, and it requires neither the assistance of Mdj1p and Yge1p nor ATP hydrolysis. We have observed a similar two-pathway reactivation of heat-denatured luciferase by the bacterial Hsp70 and the yeast cytosolic Hsp70 systems.

Indocyanine green angiographic findings of lacquer cracks in pathologic myopia.

Lacquer cracks are thought to represent healed mechanical breaks in the retinal pigment epithelium, Bruch's membrane, and choriocapillaris complex. In this study, we analyzed the indocyanine green (ICG) angiographic features of lacquer cracks and compared them with findings using fluorescein angiography. Complete ophthalmologic examinations, fluorescein angiography, and ICG angiography were performed in 29 consecutive patients (37 eyes) with lacquer cracks. Fluorescein angiograms of the cracks revealed linear hyperfluorescence in all 37 eyes. Using ICG angiography, we observed linear hypofluorescence in all 37 eyes. In 15 of 37 eyes, the length of the hypofluorescent lesion detected by ICG angiography was longer than the hyperfluorescent lesion observed by fluorescein angiography. In 17 of 37 eyes, more lacquer cracks were observed by ICG angiography than by fluorescein angiography. These findings indicate that ICG angiography can detect the development of the lesion more precisely, and may provide useful information for diagnosing pathologic myopia.

Homologous p35 proteins of baculoviruses show distinctive anti-apoptotic activities which correlate with the apoptosis-inducing activity of each virus.

The anti-apoptotic activity of p35s from two baculoviruses, Autographa californica nucleopolyhedrovirus (AcNPV) and Bombyx mori NPV (BmNPV), was compared in mammalian cells. AcNPV p35 efficiently blocked apoptosis induced by caspase overexpression, but BmNPV p35 did so very poorly. Analysis of chimeric p35s and in vitro cleavage of wild type p35s suggest that the cleavage efficiency of p35 correlates with the blocking activity. Single amino acid substitutions of BmNPV p35 with those observed in AcNPV p35, however, resulted in significant loss of its anti-apoptotic activity. We speculate that sequences flanking the cleavage site have uniquely evolved during baculovirus evolution.

Portal-hepatic venous shunt through a portal aneurysm complicated by hepatic encephalopathy and pulmonary hypertension.

We report a rare case of portal-hepatic venous shunt through an enormous portal aneurysm complicated by pulmonary hypertension. A 66-year-old woman was admitted to our hospital for hepatic encephalopathy. Chest roentgenography revealed pulmonary hypertension. Computed tomography and ultrasound examination demonstrated a shunt between the portal and hepatic veins through an enormous portal aneurysm. The diagnoses of portal-hepatic venous shunt and pulmonary hypertension were confirmed by hepatic venous catheterization and cardiac catheterization. Pulmonary hypertension might result from the effects of vasoconstrictive agents, which should be metabolized by the liver in normal subjects, passing through the intrahepatic shunt into the lung.

Biochemical characterization of glycyrrhizin as an effective inhibitor for hyaluronidases from bovine testis.

The inhibitory effects of several anti-inflammatory agents, including glycyrrhizin (GL), on the activities of hyaluronidases (HAses) purified from bovine testes and Streptomyces were investigated in vitro. It was found that (i) GL inhibits the activity of HAse (p55) from bovine testes in a dose-dependent manner, but does not affect HAse from Streptomyces; (ii) GL was the most effective of the compounds tested on bovine testis HAse activity (50% inhibition with approx. 3 microM GL); and (iii) glycyrrhetinic acid (GA), a derivative (oGA) of GA and diglucuronic acid had no detectable effects on HAse activity at 9.0 microM. The GL-induced inhibition of HAse activity is uncompetitive for its substrates. Data are provided to support the contentions that (i) bovine testis HAse (p55) is a GL-binding protein; and (ii) GL acts as a potent inhibitor of HAse in vitro.

The long-term follow-up of a highly myopic patient with a macular vortex vein.

We followed a highly myopic patient with a macular vortex vein for 11 years. His refraction was -12D OU at age 10 years when the vein was first observed ophthalmoscopically in the left eye. Eleven years later, his myopia had progressed (4 diopters OD; 8 diopters OS), and the axial lengths were elongated (+1.8 mm OD; +2.7 mm OS). The macular vortex vein was dilated and tortuous. Indocyanine green angiography revealed the outflow route of this vein in the posterior pole, where both nasal and temporal posterior choroidal venous blood collected. Computed tomography showed that the vein left the eyeball directly and coursed along the optic nerve 5 mm posterior to the optic disc. A major collecting channel of posterior choroid outflow in some highly myopic eyes, a macular vortex vein may be formed at a relatively early age, and continue to enlarge, with elongation of axial length and progression of myopia.

[Influence of sleep apnea on nocturnal blood pressure].

In the present study, we subjected 65 patients to overnight monitoring and continuous nocturnal blood pressure measurement in order to assess the influence of sleep apnea on the circulatory system. Thirty-one patients were compared before and after surgery. The severity of sleep apnea was classified by Apnea Hypopnea Index (AHI), the duration of exposure to low-level oxygen (calculated as the desaturation time: DT), and increment of blood pressure. Before surgery, a significant correlation was noted between the DT and blood pressure changes. Therefore, this index was considered useful for assessing the influence of sleep apnea on nocturnal blood pressure. After surgery, improvement of AHI was greater than 50% in 19/31 patients (61.3%), and this result was almost the same as described in the literature. The improvements in DT and BP change were greater than 50% in 21/31 (67.7%) and 14/31 (45.2%), respectively. With regard to severity before surgery, AHI was > or = 50 and DT was > or = 40% in 10 and 18 patients, respectively. Nineteen patients had BP changes > or = 40 mmHg. After surgery, 1,5, and 2, patients, respectively, still showed these values. Thus, a beneficial effect of surgery was demonstrated.

Indocyanine green angiography of retrobulbar vascular structures in severe myopia.

PURPOSE: To evaluate angiographic findings of retrobulbar arteries and veins in severely myopic patients. METHODS: We examined 416 severely myopic eyes (213 patients) with refractive errors greater than -8.25 diopters using indocyanine green videoangiography. A control group of 74 eyes (37 patients) had refractive errors within plano +/- 3 diopters. Four severely myopic patients underwent computed tomographic angiography to identify the entire intraorbital course of retrobulbar veins. RESULTS: Of 416 severely myopic eyes, 231 (55.5%) exhibited retrobulbar arteries, which were tortuous and pulsatile behind the posterior pole of the globe. Retrobulbar arteries connected directly with choroidal arteries temporal to the macular area. In 17 of these 231 eyes, retrobulbar arteries were also observed nasal to the optic nerve head, continuous with the Zinn-Haller ring around the optic nerve head and directly connected with choroidal arteries. In 39 severely myopic eyes (31 patients), indocyanine green angiography disclosed retrobulbar veins, most of which coursed vertically just behind the posterior pole of the globe. These retrobulbar veins originated as an inferior vascular network of the inferior orbital vein and drained into the superior orbital vein in the upper orbit. CONCLUSION: Retrobulbar arteries observed in this study were temporal and nasal short posterior ciliary arteries. Only the lateral collateral vein, which was one of the collateral channels between the superior and inferior orbital veins, was visible in severely myopic eyes. Indocyanine green angiography is useful in evaluating retrobulbar vascular structure in severely myopic eyes.

Synthesis of glycyrrhizin analogues containing fluorinated beta(1-->2)-linked disaccharides.

For studies on the recognition mechanisms for Glycyrrhizin-induced biological activities, seven Glycyrrhizin analogues with 3'-, 4'-, 6'-, 3-, and 4-fluorinated 2-O-beta-D-glucopyranosyl-beta-D-glucopyranoses (1-5) and 3- and 4-fluorinated 2-O-beta-D-glucopyranuronosyl-beta-D-glucopyranoses (6 and 7) were synthesized through a stepwise glycosylation procedure. 1,2-Di-O-acetyl-4,6-di-O-benzyl-3-deoxy-3-fluoro- (13) and 1,2-di-O-acetyl-3,6-di-O-benzyl-4-deoxy-4-fluoro-D-glucopyranose (14) were employed for the first beta-glycosylation of methyl glycyrrhetate, promoted with trimethylsilyl trifluoromethanesulfonate.

Identification of intermediates in the conversion of cholesterol to pregnenolone with a reconstituted cytochrome p-450scc system: accumulation of the intermediate modulated by the adrenodoxin level.

Dihydroxycholesterol and pregnenolone were clearly detected on HPLC when 22R-hydroxycholesterol was incubated with a reconstituted P450scc system containing equimolar amounts of P450scc and adrenodoxin. The dihydroxycholesterol, which has been accepted to be an intermediate in the conversion of 22R-hydroxycholesterol to pregnenolone, accumulated when adrenodoxin was at a subsaturating level with respect to P450scc. The formation of the intermediate increased with increasing pH in the range of 7.2 to 8.1, and the ratio of the intermediate to the product, pregnenolone, increased with increasing pH. When the binding of P450scc to adrenodoxin was weakened by elevation of the ionic strength, the formation of the intermediate relative to the product increased. The apparent Km for dihydroxycholesterol at a subsaturating level of adrenodoxin was about 7 microM, in contrast to 4 microM at a saturating level of adrenodoxin, implying that the affinity of dihydroxycholesterol is lower at a subsaturating level of adrenodoxin than at a saturating one. These results suggest that a subsaturating level of adrenodoxin weakened the binding of dihydroxycholesterol to P450scc and thus the intermediate, dihydroxycholesterol, was released. An intermediate other than dihydroxycholesterol, obtained when cholesterol was used as the substrate, was identified as 22R-hydroxycholesterol by HPLC and mass spectroscopic analysis. The intermediate obtained when 22R-hydroxycholesterol was used as the substrate was identified as 20R,22R-dihydroxycholesterol by HPLC, mass, and 1H-NMR spectroscopic analyses. These results provide direct evidence that cholesterol is metabolized to pregnenolone by way of 22R-hydroxycholesterol and 20R,22R-dihydroxycholesterol by P45 scc.

Nitric oxide production by coronary conductance and resistance vessels in hypercholesterolemia patients.

NG-monomethyl-L-arginine (L-NMMA), a specific inhibitor of nitric oxide (NO) synthesis, was used to investigate the effects of inhibition of NO synthesis on the coronary conductance and resistance vessels in hypercholesterolemic patients. Acetylcholine (3 and 30 micrograms/min) was administered to 10 hypercholesterolemic and 10 control patients before and after L-NMMA (25 micromol/min) infusion. Epicardial coronary diameter was measured by quantitative angiography, and coronary blood flow (CBF) was derived from Doppler flow-velocity and coronary diameter measurements. In hypercholesterolemic patients, acetylcholine-induced dilation of epicardial arteries was attenuated, and the percentage increase in CBF caused by acetylcholine was smaller than that in control patients. L-NMMA attenuated acetylcholine-induced dilation of epicardial arteries in control patients. L-NMMA had no effect on CBF responses to acetylcholine in both patient groups. L-NMMA significantly decreased the baseline coronary diameter and CBF in both groups. These results indicated that hypercholesterolemia impaired the acetylcholine-induced dilation of the conductance and resistance coronary vessels. This impairment in the conductance vessels was dependent on NO production; that of resistance vessels was not. The basal release of NO in conductance and resistance vessels was preserved in hypercholesterolemic patients.

Flow-mediated vasodilation of human epicardial coronary arteries: effect of inhibition of nitric oxide synthesis.

OBJECTIVES: This study sought to investigate the role of nitric oxide, an endothelium-derived relaxing factor, in flow-mediated vasodilation in human epicardial coronary arteries. BACKGROUND: Endothelium-derived relaxing factors may be released from the coronary artery endothelium in response to increases in blood flow. METHODS: We studied the effect of the nitric oxide synthesis inhibitor NG-monomethyl-L-arginine (L-NMMA) on the flow-mediated vasodilation of epicardial coronary arteries in 12 patients, using quantitative angiographic and Doppler flow velocity measurements. Adenosine at 100 micrograms/min was infused into the left anterior descending coronary artery to test the dilator response of the proximal artery to increases in blood flow. Acetylcholine at 3 and 30 micrograms/min was infused into the left coronary ostium to determine endothelium-dependent vasodilation of the proximal left anterior descending artery. Adenosine and acetylcholine were infused before and after the intracoronary infusion of L-NMMA (25 mumol/min for 5 min). RESULTS: Infusion of L-NMMA caused a significant decrease in the baseline diameter of the proximal left anterior descending artery (from 2.90 +/- 0.14 to 2.74 +/- 0.13 mm [mean +/- SEM], p < 0.01). Adenosine increased coronary blood flow before and after L-NMMA (+399.5 +/- 27.5% and +511.9 +/- 33.3%, respectively). Flow-mediated vasodilation was observed in the proximal left anterior descending artery before and after L-NMMA (+9.2 +/- 1.5%, p < 0.01 and +8.6 +/- 2.1%, p < 0.01, respectively). A dose of 3 micrograms/min of acetylcholine significantly dilated the proximal left anterior descending artery before L-NMMA (+7.6 +/- 1.0%, p < 0.01), but acetylcholine-induced vasodilation was attenuated after L-NMMA (-1.8 +/- 1.0%). CONCLUSIONS: Our data suggest that nitric oxide modulates basal coronary artery tone but that mediators other than nitric oxide may be responsible for the flow-mediated vasodilation of human epicardial coronary arteries.