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.

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.

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.

Behavior of mitochondria, chloroplasts and their nuclei during the mitotic cycle in the ultramicroalga Cyanidioschyzon merolae.

The behavior of cell nuclei, mitochondria and chloroplasts was followed during the mitotic cycle in the primitive unicellular red alga Cyanidioschyzon merolae by epifluorescence microscopy after staining with 4',6-diamidino-2-phenylindole (DAPI) or 3,3'-dihexyloxacarbocyanine iodide (DIOC6), by fluorimetry using a video-intensified microscope photon-counting system (VIMPCS), and by electron microscopy. The cells were classified into five types based on differences in the shape, size, and distribution of organelles in each cell: type I, II, III, IV, and V. Types II, III, IV, and V appear in that order concomitant with a decrease in type I after the cells are subjected to darkness during synchronous culture, suggesting that the organelles divide in the following sequence: chloroplast, mitochondrion, and cell nucleus. The mitochondrial DNA (mt-DNA) and the chloroplast DNA (cp-DNA) are synthesized during stage I, while the cell-nuclear DNA (cn-DNA) is duplicated in stage II. The mitochondria- and chloroplast-nuclear divisions are completed in stage IV. The mitochondrial and chloroplast divisions begin simultaneously in stage II, and chloroplast division finishes just prior to mitochondrial division. The end result is that C. merolae multiplies by binary fission. Electron microscopic examination of serial thin sections of the chloroplast clearly show that the chloroplasts in types II and III are divided by a chloroplast dividing ring (CD-ring), which is located on the cytoplasmic side of the outer envelope at the equatorial region between the daughter chloroplasts. The CD-ring may be involved in chloroplast division throughout the plant kingdom in primitive unicellular algae and higher plants.

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.

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.

Calcium-dependent slow action potentials in potassium-depolarized guinea-pig ventricular myocardium enhanced by barium ions.

Experiments were performed to investigated the mechanism by which Ba ions facilitate slow response in the myocardium. In partially depolarized guinea-pig papillary muscles, adding 0.2 mmol/l Ba to K-rich solution drastically lowered the stimulus threshold and converted the graded response to an all-or-none activity. This change was accompanied by an increase in the membrane resistance, as determined by cable analysis. When [Ca]0 was altered (0.9--7.2 mmol/l) in the presence of 0.2 mmol/l Ba, the action potentials varied almost in a manner expected for a Ca-electrode, and there were concomitant increases in the twitch tension. The electrical and mechanical activities of muscles treated with 1 mmol/l Ba were also strongly Ca-dependent. At normal [Ca]0, increasing [Ba]0 from 0.2--1 mmol/l only slightly enhanced the action potentials without any appreciable change in the peak twitch tension. When [Ca]0 was lowered to 0.2 mmol/l, elevation of [Ba]0 to over 0.5 mmol/l restored the action potentials, and the resting tension increased. These results suggest that the facilitatory action of the low concentration of Ba on the Ca-dependent slow action potentials is due to a reduction in the membrane shunting conductance and not to a development of any sizeable inward Ba current. However, Ba-dependent action potentials may be generated under conditions of Ca-deficiency. Isoprenaline (0.5--5 x 10(-7) mol/l) induced an automatic activity in the Ba-treated muscles and this phenomenon is probably related to both the drug-induced increase in Ca-permeability and the Ba-induced decrease in shunting conductance. Thus Ba ions appear to be a feasible tool for studies on the myocardial slow channel.

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.

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.

Intestinal pseudo-obstruction due to dialysis amyloidosis.

Marked gastric dilatation and the carpal tunnel syndrome developed concurrently in a 58-year-old man treated with hemodialysis for 14 years. Despite extensive examinations, no attributable organic lesion was demonstrated in the gastrointestinal tract. However, amyloid deposition, which was immunohistochemically identified as beta-2 microglobulin, was demonstrated in the stomach and the synovia excised from the right carpal tunnel. Intestinal pseudo-obstruction due to dialysis amyloidosis was considered to be the cause of the gastric dilatation. There have been a few recently reported cases with dialysis amyloidosis in which extra-articular amyloid deposition resulted in overt clinical problems. The present case provides additional evidence that dialysis amyloidosis can develop serious extra-articular complications; intestinal pseudo-obstruction is a very rare gastrointestinal manifestation.

Total synthesis of VIM-2 ganglioside isolated from human chronic myelogenous leukemia cells.

A total synthesis of the tumor-associated glycolipid antigen, VIM-2, is described [2]. Phenyl 2,3,4-tri-O-benzoyl-6-O-benzyl-beta-D-galactopyranosyl-(1-->4)-6-O-benzy l-2- deoxy-2-phthalimido-1-thio-beta-D-glucopyranoside (7), a key intermediate prepared by condensation of phenyl 6-O-benzyl-2-deoxy-2-phthalimido-1-thio-beta-D-glucopyranoside (6) and 2,3,4-tri-O-benzoyl-6-O-benzyl-alpha-D-galactopyranosyl bromide (5), was glycosylated with methyl 2,3,4-tri-O-benzyl-1-thio-beta-L-fucopyranoside (8) to give the trisaccharide donor 9, which, on coupling with 2-(trimethylsilyl)ethyl 2,4,6-tri-O-benzyl-beta-D-galactopyranosyl-(1-->4)-2,3,6-tri-O-benzyl-be ta-D- glucopyranoside (10), afforded the pentasaccharide 11. The regioselective glycosylation of 12 (derived by O-debenzoylation of 11) with 7 gave the heptasaccharide 13, which was converted by treatment with hydrazine monohydrate and subsequent N-acetylation into the hexasaccharide acceptor 14. The stereo- and regio-selective glycosylation of 14 with methyl (phenyl 5-acetamido-4,7,8,9-O-benzoyl-3,5-dideoxy-2-thio-D-glycero-beta-D-galact o-2- nonulopyranosid)onate (16) gave the desired octasaccharide 18. Hydrogenolytic removal of the benzyl groups in 18 and successive O-acetylation, removal of the 2-(trimethylsilyl)ethyl group, and treatment with trichloroacetonitrile gave the alpha-trichloro-acetimidate 21, which was then coupled with (2S,3R,4E)-2-azido-3-O-(tert-butyldiphenylsilyl)-4-octade cene-1,3-diol (22) to give 23. Compound 23 was transformed, via selective reduction of the azido group, N-introduction of octadecanoic acid, O-desilylation, O-deacylation, and saponification of the methyl ester group, into the title VIM-2 ganglioside 26.

High resolution ultrasound of the chest wall.

To study the detailed normal ultrasonic anatomy of the pleura and chest wall, high resolution (7.5 MHz) ultrasonograms were obtained from cadaver chest wall specimens and compared with thin section computed tomograms and anatomical specimens. Ultrasonograms show three layers of the intercostal muscles (internal, external and innermost), covered by the "echogenic pleural line." The "echogenic pleural line" is caused by composite echoes from the inner parietal pleura, and the outer endothoracic fascia, with the fatty tissue covering both sides of the fascia, which are located deep to the chest wall muscles. On ultrasonograms, the subpleural fat tissue, when abundant, appeared as an apron-like structure hanging down from the inner surface of the rib (subpleural fat pad), or diffuse fat accumulation mimicking the pleural thickening.

Effects of stimulus rate, temperature, and external ion compositions on hypertonicity-induced changes in the action potential in guinea-pig ventricular muscle.

When the guinea-pig ventricular muscle was exposed to hypertonic solution, duration of the action potential (APD) initially increased and then progressively decreased. To investigate the mechanism underlying this phenomenon, the preparations were perfused with the hypertonic solution under various experimental conditions. At low stimulus rates (0.1-1/min), the initial prolongation of APD was prominent while the later shortening of APD developed slowly, thereby suggesting that the effect of hypertonic solution on APD is dependent on the muscle activity. A pronounced shortening of APD occurred when the osmotic challenge was made at reduced [K]o (2 mM-K), at reduced [Na]o (50%-Na), and at elevated [Ca]o (5.4 mM-Ca). Lowering the temperature from 36 to 26-27 degrees C nearly abolished the development of APD shortening. The resting potential and the maximum rate of rise (Vmax) of the action potential changed little even when APD was rapidly shortened during the osmotic challenge. Thus, the mechanism of APD shortening is though to be independent of the factors determining the resting potential and Vmax. Mn ions (1-2 mM), verapamil (5-10 microM), and Ba ions (0.05-0.2 mM) had no effect on the hypertonicity-induced changes in APD. In partially depolarized preparations, hypertonic solution increased the duration of Ca-dependent slow action potentials without producing any parallel increase in their upstroke velocity. It is postulated that the initial prolongation of APD in response to hypertonicity is a direct result of cell dehydration. The later shortening of APD is probably derived from certain changes in the cell condition which developed secondarily to the cell dehydration. In these changes in APD, the slow Ca current seems to play a small role.

Effects of hypertonic solution on action potential and input resistance in the guinea-pig ventricular muscle.

The effects of hypertonic solution on action potential and input resistance were studied using the guinea-pig ventricular muscle. Hypertonic solutions containing excess (150 and 300 mOsm/liter) sucrose, glucose, NaCl, Na2SO4, or LiCl produced a prolongation of action potential duration (APD) followed by a gradual shortening. These solutions increased the input resistance of the preparation. The prolongation of APD began immediately after the onset of perfusion with hypertonic solution and peaked within about 10 min, while the late shortening of APD developed progressively. On introducing the isotonic solution after the osmotic challenge, APD initially shortened and then gradually prolonged toward the control level. Slightly hypertonic solutions containing excess 30 or 75 mM sucrose prolonged APD, to some extent. It is concluded that the hypertonic solution exerts early and late effects on APD in guinea-pig ventricular muscle. The early effect, which is responsible for the APD prolongation, seems to be directly related to the development of cell dehydration. The hypertonic solutions produced a slight increase in the resting potential, and sucrose- and glucose-hypertonicity depressed the rising phase of the action potential. These effects can be explained by the cell dehydration and associated changes in the intracellular ion concentrations. The increase in input resistance produced by hypertonic solutions was accompanied by an enhanced spatial decay of electrotonic potentials along the muscle bundle, as determined by the two microelectrode method, suggesting that the electrical resistance of the internal current pathway increases under hypertonic conditions.

Transient increase in the slow inward current following acetylcholine removal in catecholamine-treated guinea-pig Purkinje fibers.

In voltage-clamped guinea-pig Purkinje fibers, removal of acetylcholine (ACh) in the presence of isoproterenol produced a rebound increase in the slow inward current. This effect of ACh was abolished by atropine. These results are consistent with the hypothesis that cessation of the stimulation of myocardial muscarinic receptors transiently leads to enhanced activity of the beta-receptor/slow channel system in this tissue.

Regulation of cyclic AMP-dependent Cl- channel in heart.

(1) alpha 1-Adrenoceptor stimulation inhibited the beta-adrenergic activation of Cl- current in guinea-pig ventricular cells. This alpha 1- and beta-adrenergic interaction appeared to be upstream in the cascade of adenylate cyclase activation. (2) Single Cl(-)-channel currents were recorded in cell-attached and excised outside-out patch membranes. Experiments were performed to investigate the dependence of channel behavior on the agonist concentrations.

Interaction between caffeine and adenosine on the membrane current and tension component in the bullfrog atrial muscle.

Antagonistic action of caffeine and adenosine were studied at the membrane current and tension component levels in myocardium of the bullfrog atrium, mainly under voltage clamp using the double sucrose-gap method. Caffeine (1-5 mM) produced marked augmentation of the phasic and tonic tensions as well as slow inward current (Is). At higher concentrations it elicited an increase of the delayed outward current (Ix). The augmentation of Is was principally due to increase of the limiting conductance (gs), while the activation and inactivation variables (dinfinity, finfinity) were not changed significantly. The dose-tension response curve for caffeine, which appeared sigmoidal, was notably lowered in the presence of adenosine (1-3 mM), indicating a competitive type of inhibition. Adenosine, in the presence of caffeine, exerted a narcotic-like action by inhibiting all of the membrane currents (INaf, Is, Ix and background inward current, Ib) and tension components (ICa-dependent and independent tensions). The inhibition of Is was due to a decrease of gs and no appreciable change was observed in dinfinity and finfinity. These results suggest that adenosine has a strong stabilizing action on the myocardium, especially when the heart muscle activity is accelerated by increased cyclic AMP.

Calcium channel currents in isolated guinea-pig ventricular cells superfused with Ca-free EGTA solution.

Changes in membrane currents seen in Ca-free, EGTA (1 mM)-containing Tyrode solution (EGTA Tyrode), were studied in isolated guinea-pig ventricular cells, under the voltage clamp performed with a "G omega seal" patch electrode. Application of the EGTA Tyrode (calculated [Ca]0 = 1.3 X 10(-9) M) first eliminated the usual calcium current, but induced an extra inward current within 2 min. The reversal potential of this current, as judged by the direction of the current change, was about +25 mV (without correction of a liquid junction potential of -12 mV), but above this voltage a decaying outward current was observed. The decay of these inward and outward currents during depolarization was slow, but a large, nearly time-independent component was evident. These currents, regardless of their polarity and time course, were reduced by application of verapamil (10(-5) M) and Mg (5 mM), and were inactivated by pre-depolarizations. In Na-free EGTA Tyrode, the inward current disappeared but the outward current persisted at high voltages. These results suggest that in ventricular cells, reduction of external Ca concentrations to a nanomolar range induces a Ca channel current composed of an inward current carried by Na, and an outward current, presumably carried by K ions. Because of the persistence of the apparently non-inactivating Ca channel current, the net membrane current evoked at voltages around 0 mV remained close to zero, or even inward, after the decay of the time-dependent component, which was completed within a few hundreds ms. This characteristic I-V relation was considered to be linked to the development of the long-lasting action potentials, with a plateau maintained at around 0 mV, in EGTA Tyrode.