Shikonin inhibits IgE-mediated histamine release by human basophils and Syk kinase activity.

OBJECTIVE: Shikonin, a component of the herbal medicine "Shikon", is known to suppress inflammatory reactions, but its molecular targets are not identified. This study examines the effect of shikonin on human basophil degranulation response and aims to identify its targets. MATERIALS: Human basophils in isolated leukocytes from healthy volunteers' peripheral blood; recombinant human Syk and Lyn tyrosine kinases. METHODS: Histamine release from basophils stimulated with anti-IgE antibody was analyzed fluorimetrically. Syk and Lyn kinase activities were tested in Vitro with recombinant proteins and analyzed by off-chip mobility shift assay. RESULTS: Shikonin dose-dependently inhibited the histamine release from basophils induced by anti-IgE antibody (IC50 = 2.6 +/- 1.0 microM; mean +/- SEM). A search for the target(s) of shikonin in the signal cascade of IgE-mediated activation showed that it strongly inhibits Syk (IC50 = 7.8 microM, in the recombinant kinase assay), which plays a pivotal role in the degranulation response. A less significant inhibition was found for Lyn, which phosphorylates FcepsilonRI-betagamma subunits and also Syk. CONCLUSIONS: These results indicate that the inhibition of Syk-dependent phosphorylation events might underlie the blocked histamine release from human basophils, thus contributing to the anti-inflammatory effects of shikonin.

Autophagy in embryonic erythroid cells: its role in maturation.

Yolk sac-derived embryonic erythroid cells differentiate synchronously in the peripheral blood of Syrian hamster. The stage of differentiation on day 10 of gestation is equivalent to polychromatophilic erythroblast stage and that on day 13 is equivalent to the reticulocyte stage in adult animals. The cytoplasm of embryonic erythroid cells became scant and devoid of most organelles on day 12 of gestation. In addition, there were very few non-erythroid cells in circulation before day 13. Thus the embryonic erythroid cells serve a pure and synchronous system to study the mechanisms of terminal differentiation. The number of mitochondria in the embryonic erythroid cells decreased to about 10% of the initial number during the period between day 10 and day 12 of gestation. In contrast, the frequency of autophagy of mitochondria increased 4.6-fold in the same period. The cytochrome c content of the cell decreased as the mitochondria became extinct. However, release of cytochrome c into the cytoplasm was not detectable through day 10-13 of gestation, suggesting that the mitochondria were digested within a closed compartment. Decomposed mitochondria and ferritin particles were detected in lysosomes by electron microscopy on and after day 12 of gestation, which also suggested digestion in a closed compartment. Mitochondrial ATP synthase subunit c, which is known to be a protease-refractory protein, was retained in the cells even after the disappearance of mitochondria, indicating that most of the mitochondria were not extruded from the cells. The digestion of mitochondria in autolysosomes may allow the cells to escape from rapid apoptotic cell death through concomitant removal of mitochondrial death-promoting factors such as cytochrome c.

Agonist-induced isometric contraction of smooth muscle cell-populated collagen gel fiber.

String-shaped reconstituted smooth muscle (SM) fibers were prepared in rectangular wells by thermal gelation of a mixed solution of collagen and cultured SM cells derived from guinea pig stomach. The cells in the fiber exhibited an elongated spindle shape and were aligned along the long axis. The fiber contracted in response to KCl (140 mM), norepinephrine (NE; 10(-7) M), epinephrine (10(-7) M), phenylephrine (10(-6) M), serotonin (10(-6) M), and histamine (10(-5) M), but not acetylcholine (10(-5) M). Phentolamine (10(-7) M) produced a parallel rightward shift of the NE dose-response curve. Moreover, NE-induced contraction was partially inhibited by nifedipine and completely abolished by the intracellular Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester, the myosin light chain kinase inhibitor ML-9, the Rho kinase inhibitor Y-27632, and papaverine. A [(3)H]quinuclidinyl benzilate binding study revealed that the loss of response to acetylcholine was due to the loss of muscarinic receptor expression during culture. The expression of contractile proteins in the fibers was similar to that in cultured SM cells. These results suggest that, although the fiber is not a model for fully differentiated SM, contractile mechanisms are maintained.

Expression of cofilin isoforms during development of mouse striated muscles.

Cofilin (CF) is an actin regulatory protein that plays a critical role in actin filament dynamics in a variety of cells. Two cofilin isoforms. muscle-type (M-CF) and nonmuscle-type (NM-CF) encoded by different genes, exist in mammals; in the adult, the former is predominantly expressed in muscle tissues, while the latter is distributed in various non-muscle tissues (Ono et al., 1994). In this study, we examined cofilin isoform expression during skeletal and cardiac muscle development in mice using cDNA probes and antibodies which distinguish the isoforms. We found that the expression of M-CF was initiated in terminally differentiated myogenic cells in both the myotome and limb buds. In myogenic cell cultures, its expression occurred coupled with myotube formation. NM-CF was expressed in developing skeletal and cardiac muscles but disappeared from skeletal muscle during postnatal development, while its expression persisted in the heart, even in adult mice. A similar situation was observed in the heart of other mammals. Thus, it is likely that the both cofilin isoforms are involved in the regulation of actin assembly during myofibrillogenesis. Only M-CF could be involved in actin dynamics in mature skeletal muscle, while both isoforms could be in the mature heart.

Dissection of the effects of tumor necrosis factor-alpha and class II gene polymorphisms within the MHC on murine systemic lupus erythematosus (SLE).

Gene(s) in the MHC of the NZW strain (H-2z) up-regulate(s) systemic lupus erythematosus (SLE) in (NZB x NZW) F1 mice. So far, two plausible mechanisms have been implicated: (i) unique mixed haplotype class II molecules formed in the F1 mice act as a restriction element for self-reactive T cells and (ii) a unique polymorphism in the H-2-linked NZW tumor necrosis factor (TNF)-alpha allele which down-regulates TNF-alpha is contributory. Because of the difficulty in dissecting these alleles within the H-2 complex, it has not been determined which is indeed the case. We addressed this issue by establishing three different H-2-congenic (NZB x NZW) F1 mice bearing distinct haplotypes at class II and TNF-alpha regions, i.e. (NZB x NZW) F1 (H-2d/z:A(d/u)E(d/u)TNF(d/z)), (NZB x NZW.PL) F1 (H-2(d/u):A(d/u)E(d/u)TNF(d/d)) and (NZB x NZW.H-2d) F1 (H-2(d/d):A(d/d)E(d/d)TNF(d/d)). Among these, only (NZB x NZW) F1 produced a markedly lower level of TNF-alpha, due to the unique NZW TNF-alpha allele (TNF(z)). Studies of anti-DNA antibodies and lupus nephritis revealed that, compared to (NZB x NZW) F1, the disease of (NZB x NZW.H-2d) F1 was markedly reduced. In (NZB x NZW.PL) F1, the onset of renal disease was significantly delayed, while the extent of proteinuria and renal histopathology in individuals that had developed the disease was comparable to that seen in (NZB x NZW) F1. It seems likely that both class II and TNF-alpha gene polymorphisms are functioning as H-2-linked predisposing genetic elements, and that the TNF-alpha polymorphism acts to modulate an initial process of the renal disease.

The apoptotic and nonapoptotic nature of the terminal differentiation of erythroid cells.

The morphology of erythroid cells changes dramatically during the course of their terminal differentiation. According to calculations made with cytospin preparations obtained from Syrian hamster yolk-sac-derived erythroid cells, the area of nuclei at day 10 of gestation ranges from 25 to 85 micron 2 and is reduced to 15-25 micron 2 on day 13 [K. Morioka and R. Minamikawa-Tachino, Dev. Growth Differ. 35, 569-582, 1993]. The DNA and protein contents of each nucleus also decrease during this period. Nonspecific fragmentation of DNA was detected by agarose gel electrophoresis in all samples obtained from day 10 to day 13 of gestation, while distinct ladders of DNA fragments were not detected. DNA fragmentation was also detected by an in situ DNA-end labeling (TUNEL) assay. As the terminal differentiation proceeded, gradual decreases in levels of both histone H1 and most nonhistone proteins were observed by SDS-polyacrylamide gel electrophoresis, while levels of core histones appeared to be constant. In particular, lamin B2 was almost completely lost from the nuclear matrix fraction on day 11. These results suggest that the terminal differentiation of erythroid cells and apoptosis might have common mechanisms. However, expansion of the cytoplasm during the terminal differentiation distinguishes these processes. In addition, in the erythroid terminal differentiation, nuclei never form lobules or become fragmented; no apoptotic bodies are formed, occurrence of the apoptosis-like cellular change is not sporadic but rather synchronous, and the process is slow, with at least several days being required for cell death. These characteristics are different from those of typical apoptosis. Thus, the terminal differentiation of nucleated embryonic erythroid cells exhibits both apoptotic and nonapoptotic features.

5-fluorouracil and low-dose recombinant interferon-alpha-2a in patients with hormone-refractory adenocarcinoma of the prostate.

BACKGROUND: The effectiveness of a chemotherapy regimen including 5-fluorouracil (5-FU) and recombinant interferon-alpha-2a (rIFN-alpha-2a) was evaluated in hormone-refractory prostate cancer patients. METHODS: Patients received a continuous intravenous infusion of 5-FU at 600 mg/m2/day for 5 days (D1-D5), followed by a bolus injection of 5-FU on D15 and D22. Patients received intramuscular injection of rIFN-alpha-2a at 3 million IU on D1, D3, D5, D15, and D22. This schedule was repeated every 4 weeks. RESULTS: Between 1993 and 1995, 23 patients with hormone refractory prostate cancer were enrolled in this study. Two of five patients with nodal disease exhibited partial responses according to the NPCP criteria. Fourteen of 17 patients with bone disease showed stable disease. Of 21 patients assessible for response, 9 patients had a decrease in the PSA level greater than 50% of baseline. Bone pain disappeared partially or completely in 8 of 14 patients with this symptom at entry. The median overall survival was 18 months. The associate toxicity was well tolerable. CONCLUSIONS: Combination chemotherapy of 5-FU and low dose rIFN-alpha-2a in patients with hormone-refractory prostate cancer proved feasible, and with acceptable toxicity.

A case of lupus nephritis showing good clinical course and apoptosis in glomerular cells detected by the nick end labeling method.

We report here an adult patient with lupus nephritis who had a good clinical course under long-term observation. Apoptotic bodies in the glomeruli were determined in serial renal biopsy specimens by the nick end labeling method. Apoptotic bodies in the proliferated glomerular cells were detected in the 3rd renal biopsy but not in the 2nd biopsy. The clinical activities of lupus nephritis fluctuated until the time of the 3rd renal biopsy. The 3rd renal biopsy was performed because of increased proteinuria and an increased amount of hyaline, granular and red blood cell casts, with impairment of renal function. The levels of proteinuria, creatinine clearance and serum complements were improved after the 3rd renal biopsy. It appears that apoptosis might control glomerular cell proliferation and also influence the clinical course of lupus nephritis.

Dephosphorylation of cofilin in polymorphonuclear leukocytes derived from peripheral blood.

We show that human and porcine polymorphonuclear leukocytes express significant amounts of cofilin, a low-molecular-weight actin regulatory protein, as well as profilin. Fifty percent of the cofilin in the resting state was phosphorylated and dephosphorylation occurred after activation by fMLP or TPA. The time course of the dephosphorylation induced by fMLP was very rapid, ending within 1 min, while TPA induced relatively gradual dephosphorylation over a period of 10 min. Surprisingly, okadaic acid and calyculin A, potent inhibitors specific for phosphatase, both induced dephosphorylation of cofilin. This suggests that type 1 alone or both type 1 and 2A phosphatases are involved in the maintenance of the level of phosphorylation of cofilin in resting cells. The dephosphorylation of cofilin was barely detected by in vitro phosphatase assays, which can distinguish activities of types 1, 2B, and 2C. This indicates that cofilin is not dephosphorylated by conventional phosphatases. Although the dephosphorylation of cofilin was observed in cells treated with the calcium ionophore A23187, the phosphorylation level of cofilin was restored when the cells were further incubated in the presence of EGTA. Reactivation of these cells with TPA resulted in the dephosphorylation of cofilin; fMLP activation did not lead to dephosphorylation. Furthermore, a submicromolar concentration of wortmannin, which is an inhibitor specific for phosphatidylinositol 3-kinase, completely inhibited dephosphorylation of cofilin induced by fMLP, but did not suppress TPA-induced dephosphorylation. Thus, we conclude that the dephosphorylation of cofilin is differently regulated depending on either fMLP or TPA activation.

Distribution of actin, myosin, and spectrin during enucleation in erythroid cells of hamster embryo.

Yolk-sac derived erythroblasts undergo semi-synchronous maturation and some of them enucleate in the peripheral blood of embryos. We have studied the assembly and distribution of actin, myosin, and spectrin during the enucleation of Syrian hamster embryonic erythroblasts. At day 11 of the gestation, that is just before the start of the enucleation, formation of a cytoskeletal structure consisted chiefly of particulate associations of F(filamentous)-actin was detected by the staining with rhodamine-labeled phalloidin. Stress-fiber-like structures were not observed in each differentiation stage after day 10. Distribution of myosin, actin, and spectrin was studied immunocytochemically to know the role of them in the enucleation of erythroid cells that starts at late day 11 or early day 12 in the gestation. The enucleation is preceded by the approach and the subsequent attachment of nucleus to the plasma membrane. At that time, actin and myosin are present in the cytoplasmic and cortical region of the cells. From the time when the extrusion of nucleus has started, condensation of actin and myosin was observed at the cell cortex area surrounding the extruding nucleus, and a contractile ring-like structure was infrequently observed. Spectrin was observed in the cortical region of the cells, and the change of the localization of spectrin was not observed throughout the terminal differentiation process (days 10-12) of the embryonic erythroid cells. The results show the possible involvement of a myosin-actin contractile system that appears around the extruding nucleus within the mechanism of erythroid enucleation.

[5-Fluorouracil and alpha-2a interferon in patients with hormone-refractory prostate cancer].

BACKGROUND: Hormone-refractory metastatic prostate cancer remains a disease for were limited therapeutic options are available. Therefore, the establishment of newly, more effective chemotherapy is expected. Experimental data suggest that PC-3, a human hormone refractory prostate cancer cell line, showed a 2-fold increase in 5-Fluorouracil (5FU) sensitivity in the presence of alpha-2a Interferon (IFN alpha 2a) at 100 IU/ml, compared to that without IFN alpha 2a. Based on this data, we treated 11 patients with 5FU and IFN alpha 2a in order to determine the clinical response and toxicity of this combination chemotherapy. METHODS: One course of this combination chemotherapy consisted of a continuous intravenous infusion of 5FU at 600 mg/m2/day for 5 days (D1-D5) with IFN alpha 2a 3 million units (MU) intramuscularly 3 times weekly (D1, D3, D5) followed by a bolus injection of 5FU at 600 mg/m2 and IFN alpha 2a at 3 MU/day on D15 and D22. RESULTS: Based on the Response Criteria for Prostate cancer Treatment, one of 9 patients with bony metastasis had partial response, 2 patients with nodal disease on the CT scan obtained partial response. Six of 11 patients had more than 50% decrease in post-therapy prostatic antigen levels, 3 of whom obtained complete response. Significant myelosuppression did not occur. There were no chemotherapy-related deaths. CONCLUSION: These results suggest that the combination of 5FU and IFN alpha 2a, although preliminary, is an active regimen against hormone-refractory metastatic prostate cancer.

Binding capacity of serum IgA to jacalin in patients with IgA nephropathy using jacalin-coated microplates.

Binding capacity of serum IgA to jacalin in 22 patients with IgA nephropathy, 14 patients with diffuse mesangial proliferative glomerulonephritis (non-IgA nephropathy) and 20 age-matched healthy adults was examined by enzyme-linked immunoassay (ELISA) using jacalin-coated microplates. In contrast to previous findings, the binding capacity of serum IgA to jacalin in patients with IgA nephropathy measured by ELISA using jacalin-coated microplates was significantly higher than that in healthy adults. The ratio of serum IgA levels measured by this method to those obtained by single radial immunodiffusion was significantly increased in patients with IgA nephropathy. It appeared that the capacity of serum IgA binding to jacalin was marked in these patients. It is concluded that the binding capacity of serum IgA to jacalin is not ubiquitously impaired in all patients with IgA nephropathy.

[A 78-year-old woman with rheumatoid arthritis, right hemiparesis, and renal failure].

We report a 78-year old woman with 30 years history of rheumatoid arthritis and nephrotic syndrome, who developed right hemiparesis and renal failure recently. The patient was diagnosed as having rheumatoid arthritis in 1965, and had been treated with gold -sol, steroid hormone, and non-steroidal anti-inflammatory drugs intermittently. Later on her clinical course was complicated by nephrotic syndrome, however, her renal function was well compensated. Otherwise, she was apparently doing well until October of 1988 when she had an onset of anomic aphasia; she was 73-year-old at that time. She was admitted to our hospital; a cranial CT scan at that time revealed a low density area in the left temporal region, and she was diagnosed as suffered from an atherothrombotic infarction involving the left middle cerebral artery territory. She recovered soon and was discharged for out patient follow up with ticlopidine 100 mg/day. She was doing well until December 15, 1990, when she had an acute onset of nausea, vomiting, and speech disturbance; she was admitted to our hospital for the second time. On admission, she was alert, but she had motor aphasia, right hemiparesis, and dysarthria. A cranial CT scan revealed a low density area in the left temporal region extending into adjacent frontal and parietal areas including the angular gyrus; in addition, leukoaraiosis, cortical atrophy, and ventricular dilatation were noted (Fig. 1A, B). She was treated supportively, and she showed improvement in her aphasia, however, moderate weakness remained in her right upper and lower extremities. She was discharged for out patient follow up. She was doing well until May 21, 1993, when she developed difficulty in swallowing and speech. She became unable to take foods orally and she was admitted again on May 31. On admission, she was afebrile and BP was 120/80 mmHg. General physical examination was unremarkable except for pitting edema and multiple contracture of her joints. On neurologic examination, she was alert but appeared to have aphasia and dementia; she could utter only a few simple words, and was able to understand only simple questions.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of thyroid hormone on developmental transition of myosin light chains during flounder metamorphosis.

Thyroidal control of the transition of myosin isoforms during flounder metamorphosis was examined by the administration of either only thiourea (TU), a potent inhibitor of thyroid hormone synthesis, or thyroxine (T4) together with TU into premetamorphic larvae. Immersion of premetamorphic larvae in 400 microM of TU inhibited the appearance of the adult-type DTNB (5,5'-dithio-bis-nitrobenzoic acid) light chain, LC2, whereas administration of T4 with TU induced a precocious appearance of LC2 and decreased the relative amount of the larval-type DTNB light chain, LC2*. TU was administered into juveniles just after completion of metamorphosis. The treatment did not affect the composition of the myosin light chains. These results suggest that thyroid hormone irreversibly turns on the switch for transition of the DTNB light chains from larval to adult type during metamorphosis of the flounder.

A case of thin basement membrane syndrome associated with idiopathic membranous nephropathy.

A 59-year-old woman has had persistent microscopic hematuria for 10 years. A few days after the onset of upper respiratory tract infection, edema and severe proteinuria appeared. In renal biopsy, an electron micrograph revealed electron dense deposits in the subepithelial regions with diffuse thinning of the glomerular basement membrane. These glomerular findings were compatible with thin basement membrane syndrome associated with membranous nephropathy. On the basis of our review of the literature, the association of these two diseases seems to be very rare.

Effect of monoclonal antibody CD4 on expression of intercellular adhesion molecule 1 in renal tissues of ddY mice: a spontaneous animal model of IgA nephropathy.

Immunofluorescence studies were performed to determine whether the expression of intercellular adhesion molecule 1 (ICAM-1) in ddY mice, a model for IgA nephropathy (Berger's disease), is influenced by treatment with a rat monoclonal antibody to murine CD4 molecules. The ddY mice were initially treated with intravenous injections, followed by weekly intraperitoneal injections of monoclonal antibody CD4. Using immunofluorescence, the mean intensity of IgA deposits in renal glomerular mesangial areas and capillary walls of the treated ddY mice was significantly lower than that in saline-treated control ddY mice of comparable ages. Marked expression of ICAM-1 was observed in glomerular capillary walls and mesangial areas in both control and treated ddY mice aged 40 and 70 weeks. The glomerular mesangial expansion in the treated ddY mice was milder than that found in the control ddY mice. No significant difference in glomerular cell proliferation between the treated and control ddY mice was observed. Although the infiltration of CD4+ T cells in glomeruli was slightly decreased after the treatment, that of CD8+ T cells and macrophages/monocytes was marked in both control and treated ddY mice aged 40 and 70 weeks. Thus, it appears that CD4+ T cells modulate the amount of IgA deposits in glomeruli, and other factors may be involved in the expression of ICAM-1 in glomeruli of IgA nephropathy in ddY mice.

Detection of glycosylated protein in renal tissues and dermal vascular vessels in the microalbuminuric stage of diabetic nephropathy using the nitro blue tetrazolium reaction.

Detection of glycosylated protein in renal and dermal tissues was performed in patients in the microalbuminuric stage of diabetic nephropathy using the nitro blue tetrazolium (NBT) reaction. The intensity of NBT staining in glomeruli and dermal vascular vessels was marked in the microalbuminuric stage as well as in the overtly proteinuric stage. The NBT staining in the renal and dermal vascular walls in both stages was significantly stronger than in the samples of control autopsy patients. It appeared that nonenzymatic glycosylation in various tissues, i.e. kidneys and dermal vascular vessels, had already progressed in the microalbuminuric stage in patients with diabetic nephropathy.

IgA deposits might not influence the production of extracellular matrix in glomeruli of ddY mice, a spontaneous animal model for IgA nephropathy.

Immunofluorescence studies were carried out to determine whether the expression of extracellular matrix (ECM) in glomeruli of ddY mice, a model for IgA nephropathy (Berger's disease), is influenced by treatment with a rat monoclonal antibody to murine CD4 molecules (mAb CD4). This mAb CD4 showed a selective decrease in the number of CD4+ T cells in the peripheral blood of ddY mice as described previously. The ddY mice were initially treated with intravenous injections, followed by weekly intraperitoneal injections of mAb CD4. In immunofluorescence, the mean intensity of IgA deposits in the renal glomerular mesangial areas and capillary walls of the treated ddY mice was significantly lower than that in saline-treated control mice at comparable ages. There was no significant difference in the distribution or intensity of ECM components, i.e. type IV collagen, fibronectin and heparan sulfate proteoglycan, in glomeruli between the mAB CD4-treated and the control ddY mice. In light microscopy, mesangial expansion in the treated ddY mice was milder than that found in the saline control mice. No significant differences in the average number of intraglomerular cells, levels of serum IgA and urinary protein between the treated and control ddY mice were observed. Thus, it appears that although CD4+ T cells modulate the amounts of glomerular IgA deposits, other factors may be involved in the expression of ECM in glomeruli of IgA nephropathy in ddY mice.