Treatment with cyclosporine A improves SLE disease activity of Japanese patients with diffuse proliferative lupus nephritis.

AIMS: Cyclosporine A (CyA), a representative calcineurin inhibitor, may be useful for the treatment of lupus nephritis. In contrast to knowledge about its strong effects against proteinuria, however, there is little information about the beneficial effects of CyA against clinical disease activity of diffuse proliferative lupus nephritis. METHODS: To elucidate this issue, we investigated the effects of low-dose CyA treatment (< 2.5 mg/kg/d) in 11 Japanese adult patients (1 male, 10 female) with uncontrolled diffuse proliferative lupus nephritis with severe clinical SLE disease activity. RESULTS: In addition to amelioration of the proteinuric state, the clinical SLE disease activities, estimated by serological markers and the SLE disease activity index (SLEDAI), were significantly improved in all patients within 1 month. The required amounts of corticosteroid were decreased in these patients. These favorable effects continued for 2 y without serious adverse effects. Kidney function was not changed in the patients with satisfactory kidney function prior to CyA therapy (serum creatinine < 1.1 mg/dl, and eGFR > 45 ml/ min/1.73 m2). CONCLUSION: The current study results suggest that low-dose CyA treatment could ameliorate the severe clinical SLE disease activity as well as improve proteinuria in Japanese patients with diffuse proliferative lupus nephritis. This treatment would be safe and useful for SLE patients with satisfactory kidney function.

alpha-Adrenoceptor-mediated depletion of phosphatidylinositol 4, 5-bisphosphate inhibits activation of volume-regulated anion channels in mouse ventricular myocytes.

BACKGROUND AND PURPOSE: Volume-regulated anion channels (VRACs) play an important role in cell-volume regulation. alpha(1)-Adrenoceptor stimulation by phenylephrine (PE) suppressed the hypotonic activation of VRAC current in mouse ventricular cells and regulatory volume decrease (RVD) was also absent in PE-treated cells. We examined whether the effects of alpha(1)-adrenoceptor stimuli on VRAC current were modulated by phosphatidylinositol signalling. EXPERIMENTAL APPROACH: Whole-cell patch-clamp method was used to record the hypotonicity-induced VRAC current in mouse ventricular cells. RVD was analyzed by videomicroscopic measurement of cell images. KEY RESULTS: The attenuation of VRAC current by PE was suppressed by alpha(1A)-adrenoceptor antagonists (prazosin and WB-4101), anti-G(q) protein antibody and a specific phosphoinositide-specific phospholipase C (PLC) inhibitor (U-73122), but not by antagonists for alpha(1B)-, alpha(1D)- or beta-adrenoceptor, or protein kinase C inhibitors. The inhibition of VRAC by PE was antagonized by intracellular excess phosphatidylinositol 4,5-bisphosphate (PIP(2)), while intracellular anti-PIP(2) antibody (PIP(2) Ab) inhibited the activation of VRAC currents. When cells were loaded with phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) with or without PIP(2) Ab, PE little affected the VRAC current. Extracellular m-3M3FBS (an activator of PLC) suppressed VRAC in the absence of PE, and this effect was reversed by intracellular excess PIP(2). CONCLUSIONS AND IMPLICATIONS: Our results indicate that the stimulation of alpha(1A)-adrenoceptors by PE inhibited the activation of cardiac VRAC current via PIP(3) depletion brought about by PLC-dependent reduction of membrane PIP(2) level.

Reduction of Calponin h1 expression in human colon cancer blood vessels.

AIMS: Calponin h1 (CN) is a differentiation marker of smooth muscle cells that has been reported to be down-regulated in the blood vessels of several human tumors. In this study, we examined CN expression in blood vessels in relation to the clinical and pathological features of colon cancer tissue samples. METHODS: Fifty-six patients who had undergone colectomy for colon cancer were examined. To assess patients' disease-free survival, those who had metastasis at the time of surgical operation were excluded. Immunohistochemistry was performed by the indirect immunoperoxidase method, using serial sections made from formalin fixed and paraffin embedded tissue blocks. RESULTS: We found that the expression of vascular CN in the peripheral region of colon cancer tissues was significantly reduced in association with tumor progression, lymphatic invasion, vascular invasion and recurrence. This reduction of CN indicated not only a decrease of pericytes and/or smooth muscle cells in tumor vessels, but also the immaturity of those cells, since CN down-regulation occurred even in alpha-smooth muscle actin-positive cells. The down-regulation of CN in vessels in the peripheral region of tumor tissues was inversely associated with the expression of VEGF (vascular endothelial growth factor), seemingly advantageous to angiogenesis. CONCLUSION: The down-regulation of CN expression in colon cancer vasculature evaluated by immunohistochemistry may be useful in conjunction with conventional staging procedures to predict more reliable outcome and to select therapeutic treatment.

A heteroplasmic mitochondrial DNA 3310 mutation in the ND1 gene in a patient with type 2 diabetes, hypertrophic cardiomyopathy, and mental retardation.

A mentally retarded 57-year-old Japanese man with maternally-inherited type 2 diabetes was found to have hypertrophic cardiomyopathy (HCM) that was associated with pathological changes in the myocardial mitochondria. The mitochondrial DNA (mtDNA) of this patient was examined and a C3310 T mutation was found in the ND1 gene, which resulted in the substitution of serine for proline. The normal 3310 mtDNA band could not be detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in mtDNA from his myocardium, pancreas, cerebral tissue, skeletal muscle, and lymphocytes. However two clones sequenced from his pancreatic tissue did not show this C3310 T mutation while forty-eight did. Mitochondria isolated from the lymphocytes of his two sisters also had this mutation. mtDNA point mutations in the ND1 gene region reported thus far have been mostly homoplasmic. However, the C3310 T point mutation that was found in this patient was heteroplasmic, which is a high level of mutation and may represent the pathogenic gene that was responsible for causing mitochondrial disease.

Younger onset myeloperoxidase-specific antineutrophil cytoplasmic antibody- (MPO-ANCA) related glomerulonephritis accompanied with nephrotic syndrome.

It is known that nephrotic syndrome rarely accompanies myeloperoxidase-specific antineutrophil cytoplasmic antibody- (MPO-ANCA) related glomerulonephritis. We present a case of younger onset MPO-ANCA-related glomerulonephritis accompanied with nephrotic syndrome in a female patient. It was diagnosed through the renal biopsy and the detection of a high titer of MPO-ANCA and steroid therapy (intravenous steroid pulse therapy and oral administration), anticoagulant therapy and antiplatelet therapy were initiated. Since her nephrotic syndrome persisted in spite of the decrease of MPO-ANCA, we conducted a second renal biopsy. We found active necrotizing crescentic glomerulonephritis with a small deposition of immunoglobulin and fibrinogen on the glomeruli. To suppress her disease activity, we administered second steroid-pulse therapy and MPO-ANCA titer disappeared. However, as her nephrotic syndrome, which was accompanied by severe hyperlipidemia, persisted, we tried to treat her using low-density lipoprotein (LDL) apheresis. It was effective temporarily, but she finally fell into end-stage renal failure. We discuss here the possibility of double nephropathy by considering her clinical and renal pathologic features.

Reduced expression of actin-binding proteins, h-caldesmon and calponin h1, in the vascular smooth muscle inside melanoma lesions: an adverse prognostic factor for malignant melanoma.

BACKGROUND: The structural integrity of the blood vessels such as small arteries and veins is studied less frequently in malignant tumours than is angiogenesis. Objectives To clarify the characteristics of small arteries and small veins of melanoma lesions. METHODS: We immunohistochemically investigated various types of melanocytic tumours using antibodies specific for endothelial and vascular smooth muscle cells, and analysed the relationship between the expression of these molecules in the blood vessels and the biological characteristics of the tumours. Formalin-fixed, paraffin-embedded sections of 15 cases of benign melanocytic tumours and 64 cases of malignant melanomas were investigated. RESULTS: Significant suppression of expression of h-caldesmon (h-CD) and calponin h1 (CNh1) was observed in the blood vessels of malignant melanomas compared with both benign melanocytic tumours and normal tissues. In particular, the level of h-CD expression was inversely correlated with the frequency of metastasis and positively correlated with the survival rate in patients with malignant melanoma. CONCLUSIONS: These findings suggest that alterations of the tumour vessels are an important factor for the prognosis of malignant melanoma, and that suppression of h-CD and CNh1 in the blood vessels in malignant melanoma reflects a structural fragility of the vessels, leading to their easy penetration by tumour cells. Defective expression of these molecules is likely to be an important marker for metastatic potential and for poor prognosis of melanoma.

A case of mixed membranous nephropathy and purpura nephritis.

We report the case of a 71-year-old man with mixed glomerular lesions, membranous and necrotizing changes. The patient had abdominal pain and purpurat on the extremities and trunk, followed by melena, and after admission to hospital, proteinuria and occult blood were noted. Laboratory findings were negative for autoimmune disease and viral hepatitis. Renal biopsy showed segmental necrotizing changes and mesangial proliferation with spike formation. Immunofluorescence revealed a granular deposition of IgA predominantly in the mesangial area in contrast to the granular IgG deposition along the glomerular capillary loops. Moreover, electron-microscopically, mesangial as well as subepithelial electron-dense deposits were observed. These data suggest that the patient had 2 distinct types of glomerulonephritis simultaneously: idiopathic membranous nephropathy and purpura nephritis.

Calponin h1 induced a flattened morphology and suppressed the growth of human fibrosarcoma HT1080 cells.

Calponin h1 (CNh1) is an actin-binding protein that is expressed mainly in smooth muscle cells and is known to regulate smooth muscle contraction. Recently, re-expression of CNh1 in leiomyosarcoma cell lines is reported to suppress cell proliferation and tumorigenicity. However, little is known about the associated cellular structural and functional changes. Since CNh1 is also detected in normal fibroblasts, we hypothesised that CNh1 would also inhibit cell proliferation of the fibrosarcoma cells, HT1080, in which CNh1 is suppressed. An expression vector of human CNh1 complementary DNA was transfected into human HT1080 cells by a calcium-phosphate precipitation method. CNh1-transfected cells exhibited a flattened morphology with organised actin filaments, a significant decrease in cell motility and enhancement in adhesion to fibronectin in association with an increase in integrin alpha5beta1 expression. Anchorage-independent growth and tumorigenicity in nude mice were suppressed in the CNh1-transfected cells. Our results suggest that CNh1 may have a role as a tumour suppressor in human fibrosarcoma by influencing cytoskeletal activities.

Side chain effect on ion channel characters of Aib rich peptides.

As models of ion channel proteins and naturally occurring pore-forming peptides, we designed a series of Aib rich peptides [Ac-(Aib-Xxx-Aib-Ala)(5)-NH(2) (Xxx = Lys, Glu, Ser, and Gly: BXBA-20)] to investigate the effects of the side chains of the amino acid residues Lys, Glu, Ser, and Gly on the conformation and electrophysiological properties of ion channels. The conformation of peptides and their affinity for phospholipid membranes were evaluated by CD spectroscopy. Patch-clamp experiments revealed that all BXBA-20 peptides form ion channels in DPhPC bilayers exhibiting clearly resolved transitions between the open and closed states. The channel forming frequency was in the order BKBA-20>BEBA-20>BSBA-20>BGBA-20. In the case of BKBA-20 and BEBA-20, the self-assembled conductive oligomers expressed homogeneous and voltage-independent single channel conductances. In contrast, heterogeneous conductance was observed in BSBA-20 and BGBA-20 ion channels under similar experimental conditions. From these results, we conclude that peptides with a high degree of helical conformation, high amphipathicity, high affinity for lipid membranes, and self-associating characters in vesicles are most suitable for inducing ion channels with a high frequency of occurrence. Moreover, BEBA-20, BSBA-20, and BGBA-20 channels were cation-selective, whereas the BKBA-20 channel was non-selective.

Structural fragility of blood vessels and peritoneum in calponin h1-deficient mice, resulting in an increase in hematogenous metastasis and peritoneal dissemination of malignant tumor cells.

We have observed weak expression of calponin h1, which stabilizes the actin filament system, in blood vessels within human malignant tumors. This observation suggested that because of a deficiency in stabilization by calponin h1, the structure of blood vessels in malignant tumors is fragile compared with blood vessels in normal tissues. We therefore generated calponin h1-deficient (CN(-/-)) mice to examine the effect of calponin h1 on the integrity of the barrier system in blood vessels against cancer metastasis. The CN(-/-) mice exhibited morphological fragility of the tissues, including the uterus and blood vessels. In particular, we frequently observed bleeding into the surrounding tissue from blood vessels of the ocular fundus in CN(-/-) mice. In addition, mesothelial cells, which usually express calponin h1 in normal (CN(+/+)) mice, were retracted in the CN(-/-) mice. When fluorescein was injected i.v. into mice, the CN(-/-) mice exhibited a greater and more rapid leakage of fluorescein from the blood vessels of the ocular fundus compared with the CN(+/+) mice. In the CN(-/-) mice receiving i.v. inoculations of B16 melanoma cells, significantly more metastatic nodules were formed in the lung than in the CN(+/+) mice. When B16 melanoma cells were injected i.p., the severity of peritonitis carcinomatosa was greater in CN(-/-) than in CN(+/+) mice. These results indicate that calponin h1 plays an important role in the regulation of the integrity of the blood vessels and peritoneum, which in turn is an important factor influencing the frequency of cancer metastasis. The CN(-/-) mice, which exhibit fragile blood vessels and peritoneum, could serve as sensitive and useful host models to investigate cancer metastasis.

Solid-Phase synthesis of a library constructed of aromatic phosphate, long alkyl chains and tryptophane components, and identification of potent dipeptide telomerase inhibitors.

Telomerase inhibitors are expected as a new candidate of therapeutic agents for cancer. Recently, we have found novel inhibitors based on the bisindole skeleton. In this study, solid-phase synthesis was applied to construct a library of inhibitors having aromatic phosphate, long alkyl chain and tryptophane components, from which a D,D-ditryptophane derivative has been identified as a new potent telomerase inhibitor with IC(50) values of 0.3 microM. A hypothetical binding model for the new inhibitors has been proposed based on the structure-activity relationship.

Changes in cell volume induced by activation of the cyclic amp-dependent chloride channel in guinea-pig cardiac myocytes.

The effects of the activation of cyclic AMP-dependent Cl- current (ICl,cAMP) on cell volume were studied at various [K+]o under isosmotic conditions in guinea-pig ventricular myocytes. The area of the cell image obtained with videomicroscopy was used as an index of cell volume. I(Cl,cAMP) was activated by adrenaline (5.5 microM). Measurements of the membrane potential (Vm) were performed by the gramicidin-perforated patch-clamp method. At 5.4 mM [K+]o with low [Cl-]o, where Vm was negative to the predicted equilibrium potential of Cl- (ECl), adrenaline sizably decreased the cell area. At high [K+]o with normal [Cl-]o, where Vm was positive to ECl, adrenaline increased the cell area; at 145.4 mM [K+]o the cell area was increased to 110% of control on average (n = 22). The cells swollen in this manner shrank when [Cl-]o was reduced to a low level in the presence of adrenaline. The results indicate that the induction of Cl- influxes (outward I(Cl,cAMP)) or effluxes (inward I(Cl,cAMP)) can lead to a cell swelling or shrinkage, respectively. The addition of BaCl2 (1 mm), a blocker of K+ channels, attenuated the adrenaline-dependent cell swelling, supporting the view that Cl- fluxes must be accompanied by cofluxes of K+ ions to affect the cell volume. The adrenaline-dependent cell swelling was inhibited by antagonizing beta-adrenergic stimulation with acetylcholine or by blocking I(Cl,cAMP) channels with glibenclamide, confirming the involvement of I(Cl,cAMP) in the adrenaline response. The results show that the activation of I(Cl,cAMP) can shrink or inflate the cardiac cells under isosmotic conditions, depending on Vm and ECl.

Development of novel telomerase inhibitors based on a bisindole unit.

Telomerase is the enzyme that elongates telomere repeat at the ends of a chromosome. As high telomerase activity is observed in most cancer cells, inhibitors of human telomerase have been expected as new chemotherapeutic agents for cancer. We describe here the discovery of novel inhibitors with IC50 values in the submicromolar range. The structure of the novel inhibitors will be useful as a scaffold for construction of the library in the search for telomerase inhibitors.

CyA-mediated renal interstitial and vascular lesions in the rat under low-sodium diet.

Nephrotoxicity of CyA was analyzed histologically in rats fed a low-sodium diet. CyA was subcutaneously administered daily at a dose of 15 mg/kg for 10 or 35 days with or without prior uninephrectomy (UNT) in male Sprague-Dawley rats receiving a low-sodium diet (0.03% sodium). CyA-administered rats showed impaired renal function as well as tubulo-interstitial lesions, such as edema, tubular basement membrane changes, and tubular atrophy, in the cortex, especially in the subcapsular portion, within 10 days. On day 35, the tubulo-interstitial lesions were advanced with mild focal interstitial fibrosis. These lesions were mild in the UNT group compared to the non-UNT group. Immunohistochemically, CyA treatment caused an increase in number of renin-positive cells in the afferent arteriolar wall at juxtaglomerular area. These cells lost the expression of calponin, which is a marker of mature smooth muscle cells. In addition, in afferent arterioles and interlobular arteries, electron-dense fibrous bodies were found in the smooth muscle cells on days 10 and 35. Immunoelectron microscopically, these bodies showed scattered positive staining for calponin and alpha-actinin, were negative or only peripherally positive for alpha-SMA and vimentin, and were completely negative for desmin. This study revealed that CyA could cause interstitial lesions starting in the subcapsular portion of the renal cortex and vascular lesions of the preglomerular artery. Increases in number of renin granules and formation of cytoplasmic fibrous bodies in smooth muscle cells could be the forerunner of severe arteriolar wall damage.

Nodular lung disease with five year survival and unilateral pleural effusion in AL amyloidosis.

A 67-year-old female patient with biopsy proven AL systemic amyloidosis developed rapidly progressive dyspnea. Chest roentgenogram and CT scan revealed a large right pleural effusion in addition to nodular lesions with bilateral hilar lymphadenopathy. The patient's serum showed IgG lambda type monoclonal gammopathy and she also had Bence Jones proteinuria. The pleural effusion was an exudate that contained many mononuclear cells and a high concentration of protein. Cardiac function was not seriously disturbed. Except for amyloidosis, no other causes for the severe pleural effusion were found. This patient was treated with chemical pleurodesis using Picibanil and a low dose of prednisolone. Eighteen months after this treatment, her right pleural effusion did not recur. Bronchopulmonary tissues are known to be frequently involved by AL systemic amyloidosis, but a nodular pattern of pulmonary amyloid deposition and a unilateral large pleural effusion are rare clinical manifestations in this disease.

Sarcoidosis with membranous nephropathy and granulomatous interstitial nephritis.

A 49-year-old woman, who had been diagnosed as sarcoidosis based on bilateral hilar lymphadenopathy and lung biopsy, presented increased serum creatinine and calcium concentrations. Renal biopsy showed the presence of interstitial nephritis with non-caseating epithelioid granuloma and focal membranous transformation. Therapy with prednisolone was effective in normalizing serum creatinine, serum calcium, serum angiotensin converting enzyme, and urine beta2 microglobulin, but these abnormalities reappeared after rapid withdrawal of prednisolone. This is a rare case of sarcoidosis manifested by both membranous nephropathy and granulomatous interstitial nephritis, and indicates the necessity of long-term treatment of corticosteroid.

Molecular mechanism for pore-formation in lipid membranes by the hemolytic lectin CEL-III from marine invertebrate Cucumaria echinata.

The pore-forming activity of CEL-III, a Gal/GalNAc specific lectin from the Holothuroidea Cucumaria echinata, was examined using artificial lipid membranes as a model system of erythrocyte membrane. The carboxyfluorescein (CF)-leakage studies clearly indicated that CEL-III induced the formation of pores in the dipalmitoyl phosphatidyl choline (DPPC)-lactosyl ceramide (LacCer) liposomes effectively but not in the DPPC-glucosyl ceramide (GlcCer) liposomes or DPPC liposomes. Such a leakage of CF was strongly inhibited by lactose, a potent inhibitor of CEL-III, suggesting that the leakage is mediated through the specific binding of CEL-III to the carbohydrate chains on the surface of the liposomes. The leakage of CF from the DPPC-lactosyl ceramide liposomes was pH-dependent, and it increased with increasing pH. The immunoblotting analysis and circular dichroism data indicated that upon interaction with liposomes, CEL-III associated to form an oligomer concomitantly with a marked conformational change. Furthermore, channel measurements showed that CEL-III has an ability to form small ion channels in the planar lipid bilayers consisting of diphytanoylphosphatidylcholine and human globoside (Gb4Cer)/LacCer.

On the mechanism of the enhancement of delayed rectifier K+ current by extracellular ATP in guinea-pig ventricular myocytes.

The effects of extracellular adenosine 5'-triphosphate (ATP) on the delayed rectifier K+ current (IK) were studied in guinea-pig ventricular myocytes using the whole-cell voltage-clamp technique. ATP increased IK concentration dependently with a concentration eliciting a half-maximal response of 1.86 microM and a maximal increase of about 1.8-fold. The enhancement of IK developed slowly, the effect reaching a maximum in about 1.6 min after application of ATP. The rank order of agonist potency in enhancing IK was 2-methylthio-ATP>/= ATP>>alpha,beta-methylene-ATP. The ATP response was attenuated in guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS)- loaded cells, but was not affected by pertussis toxin (PTX)-pre-treatment, indicating that a PTX-insensitive G protein is involved in the response. These features are consistent with operation of P2Y-type purinoceptors. ATP produced a further increase in IK stimulated maximally either by isoprenaline (1 microM) through protein kinase A (PKA) or by 12-O-tetradecanoylphorbol 13-acetate (TPA, 100 nM) through protein kinase C (PKC), while 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7, 10 microM) did not affect the ATP response, suggesting that PKA and PKC do not mediate the response. ATP irreversibly enhanced IK in cells loaded with adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS, 5 mM) or okadaic acid (10 microM), a phosphatase inhibitor, suggesting that a phosphorylation step is present after the receptor stimulation. Genistein, an inhibitor of tyrosine phosphorylation, suppressed the ATP response significantly, while daidzein, an inactive analogue of genistein, had little effect on it, although both genistein or daidzein alone decreased IK. It is hypothesized that tyrosine phosphorylation plays a role in the signalling pathway involved in the enhancement of cardiac IK by P2Y-purinergic stimulation.

Enzyme level of enterococcal F1Fo-ATPase is regulated by pH at the step of assembly.

The amount of F1Fo-ATPase in Enterococcus hirae (formerly Streptococcus faecalis) increases when the cytoplasmic pH is lowered below 7.6, and protons are extruded to maintain the cytoplasmic pH at around 7.6. In the present study, we found that the transcriptional activity of the F1Fo-ATPase operon was not regulated by pH. The synthesis of F1 subunits was increased 1.65 +/- 0.12-fold by the acidification of medium from pH 8.0 to pH 5.3. Western-blot analysis showed that there were F1 subunits in the cytoplasm, and the number of alpha plus beta subunits in the cytoplasm was 50% of the total number of the subunits in cells growing at pH 8.0. This decreased to 22% after shifting the medium pH to 5.3, with a concomitant 5.1-fold increase in the level of membrane-bound F1Fo-ATPase. The cytoplasmic F1 subunits were shown to be degraded, and Fo subunits not assembled into the intact F1Fo complex were suggested to be digested. These data suggest that regulation of the enzyme level of F1Fo-ATPase by the intracellular pH takes place mainly at the step of enzyme assembly from its subunits.