HSpin1, a transmembrane protein interacting with Bcl-2/Bcl-xL, induces a caspase-independent autophagic cell death.

The Drosophila spinster (spin) gene product is required for programmed cell death in the nervous and reproductive systems. We have identified a human homologue of the Drosophila spin gene product (HSpin1). HSpin1 bound to Bcl-2 and apoptosis regulator Bcl-X (Bcl-xL), but not to proapoptotic members such as Bcl-2-associated X protein and Bcl-2 homologous antagonist killer, in cells treated with TNF-alpha. Exogenous expression of HSpin1 resulted in the cell death without inducing a release of cytochrome c from mitochondria. Overexpression of Bcl-xL inhibited the HSpin1-induced cell death. Interestingly, a necrosis inhibitor, pyrrolidine dithiocarbomate, but not the pancaspase inhibitors, carbobenzoxy-VAD-fluoromethyl ketone and p35, blocked the HSpin1-induced cell death. HSpin1-induced cell death increases autophagic vacuole and mature form of cathepsin D, suggesting a novel caspase-independent cell death, which is link to autophagy.

Association between actin and light chains in Chlamydomonas flagellar inner-arm dyneins.

Inner dynein arms in cilia and flagella contain actin as a subunit; however, the function of this actin is totally unknown. Here we performed chemical crosslinking experiments to examine the interaction of actin with other subunits. Six of the seven Chlamydomonas inner-arm dynein species separated by anion-exchange chromatography contain actin and either one of the two previously identified light chains, p28 and centrin, in a mutually exclusive manner. Western blotting of chemically crosslinked dyneins indicated that actin is directly associated with p28 and centrin but not with the dynein heavy chains (HCs). In contrast, p28 and centrin both appeared to interact directly with the N-terminal half of the HCs. Thus it is likely that actin is associated with the heavy chains through p28/centrin. These light chains may well function in the assembly or targeting of the inner arm to the correct axonemal location.

L-Arginine treatment may prevent tubulointerstitial nephropathy caused by germanium dioxide.

BACKGROUND: Long-term oral ingestion of germanium dioxide (GeO2) causes progressive renal failure derived from tubulointerstitial nephropathy in humans and animals. The characteristic of GeO2-induced nephropathy is the renal tissue injury persisting for a long time, even after cessation of GeO2 ingestion. However, a treatment that can suppress the long-lasting renal tissue injury has not yet been established. METHODS: Using the methods of immunohistochemistry and reverse transcription-polymerase chain reaction, we examined the expression of ED1-positive cells (macrophages/monocytes), transforming growth factor (TGF)-beta1 mRNA and protein and collagen type IV mRNA and protein in the kidneys of rats with GeO2-induced nephropathy. Concomitantly, the effects of L-arginine treatment on their expression was explored in the kidneys of rats with GeO2-induced nephropathy. RESULTS: Chronic administration of GeO2 caused tubulointerstitial nephropathy characterized by leukocyte invasion into the enlarged tubulointerstitial space in rats. The expression of ED1-positive cells, TGF-beta1 protein and collagen type IV protein was markedly increased in the tubulointerstitium of the renal cortex from rats with GeO2-induced nephropathy. Similarly, TGF-beta1 and collagen type IV mRNA were significantly enhanced in the renal cortex of rats with GeO2-induced nephropathy. A small number of tubulointerstitial cells expressing TGF-beta1 protein were also observed in the renal cortex of rats with GeO2-induced nephropathy. However, L-arginine treatment led to a parallel decrease in the expression of ED1-positive cells, TGF-beta1 mRNA and collagen type IV mRNA and protein in rats with GeO2-induced nephropathy. CONCLUSIONS: In general, collagen synthesis is driven by TGF-beta1 in the fibrotic process associated with a variety of renal disorders. TGF-beta1 is secreted by TGF-beta1 producing cells such as macrophages, fibroblasts and myofibroblasts. Thus, the present study indicates that the expression of collagen type IV may be mediated by TGF-beta1 released from invading macrophages and, to a lesser extent, released from tubulointerstitial cells, presumably fibroblasts and/or myofibroblasts in GeO2-induced nephropathy. L-Arginine treatment inhibits collagen type IV synthesis possibly by suppressing macrophage invasion and the resultant TGF-beta1 expression in this nephropathy. L-Arginine treatment may be beneficial in the prevention of tubulointerstitial fibrosis, which is considered to be the terminal stage of GeO2-induced nephropathy.

Protein binding of a DRPLA family through arginine-glutamic acid dipeptide repeats is enhanced by extended polyglutamine.

Dentatorubral-pallidoluysian atrophy (DRPLA) is one of the hereditary neurodegenerative disorders caused by expansion of CAG/glutamine repeats. To investigate the normal function of the DRPLA gene and the pathogenic mechanism of neuron death in specific areas of the brain, we isolated and analyzed a gene that shares a notable motif with DRPLA, arginine-glutamic acid (RE) dipeptide repeats. The gene isolated, designated RERE, has an open reading frame of 1566 amino acids, of which the C-terminal portion has 67% homology to DRPLA, whereas the N-terminal portion is distinctive. RERE also contains arginine-aspartic acid (RD) dipeptide repeats and putative nuclear localization signal sequences, but no polyglutamine tracts. RERE is expressed at a low level in most tissues examined. Immunoprecipitation and in vitro binding assays demonstrate that the DRPLA and RERE proteins bind each other, for which one of the RE repeats has a primary role, and extended polyglutamine enhances the binding. With engineered constructs fused with a tag, the RERE protein localized predominantly in the nucleus. Moreover, when RERE is overexpressed, the distribution of endogenous DRPLA protein alters from the diffused to the speckled pattern in the nucleus so as to co-localize with RERE. More RERE protein is recruited into nuclear aggregates of the DRPLA protein with extended polyglutamine than into those of pure polyglutamine. These results reveal a function for the DRPLA protein in the nucleus and the RE repeat in the protein-protein interaction.

Exon 9 mutations in the WT1 gene, without influencing KTS splice isoforms, are also responsible for Frasier syndrome.

We report new mutations in exon 9 of the WT1 gene that did not alter the ratio of +/- KTS splice isoforms in two unrelated patients with Frasier syndrome (FS). The mutation of intron 9 inducing defective alternative splicing was reported to be responsible for this syndrome. The mutations found in our cases occurred in the same exon of the WT1 gene as detected in Denys-Drash syndrome (DDS) and could not be explained by the previously proposed mechanism. The results suggest that the two syndromes originate from the same WT1 gene abnormality. From a molecular biological point of view, we concluded that the two diseases were not separable, and that FS should be included as an atypical form of DDS.

Inflammatory disease in HLA-B27 transgenic rats.

UNLABELLED: A spontaneous inflammatory disease in rats transgenic for HLA-B27 resembles the B27-associated human spondyloarthropathies. Colitis and arthritis, the two most important features, require T cells, gut bacteria, and high expression of B27 in bone marrow-derived cells. Control rats with HLA-B7 remain healthy. Most rats with HLA-Cw6 (associated with psoriasis vulgaris) remain healthy; a minority develop mild and transient disease. Rats with a mutant B27 with a Cys67-->Ser substitution resemble wild-type B27 transgenics, but with a lower prevalence of arthritis. A similar phenotype is seen in B27 rats co-expressing a viral peptide that binds B27. Disease-prone LEW but not F344 B27 rats develop high serum IgA levels concurrent with disease progression. Colitis is associated with high interferon-gamma, arthritis with high interleukin-6. Disease is similar in B27 LEW, F344, and PVG rats, but the DA background is protective. CONCLUSIONS: The spondyloarthropathy-like disease in rats is specific for HLA-B27 but does not require Cys67. Arthritis but not colitis is particularly sensitive to B27 peptide-binding specificity. Genetic background exerts a strong influence, but some phenotypic differences exist between permissive strains that do not influence disease susceptibility. The data favor a role for B27 peptide presentation in arthritis, but other mechanisms to explain the role of B27 have not been excluded.

[Home medical care from our hospital].

From April, 1996 to March, 1999, our hospital provided home medical care on a 24-hour basis for fifty patients with advanced or terminal cancer. Eventually, twenty-four patients died at home and twenty-six in the hospital. Stability of health status, the presence of willing and able caregivers, as well as a greater number of house-calls are suggested factors in facilitating a death at home. However, the patients who died in the hospital were obliged to readmit themselves until the time of death due to caregivers' reasons such as fatigue, emotional stress and/or health problems. In addition to timely availability and accessibility of respite care, psychosocial support for family caregivers by liaison nurses remains an issue to be solved in future.

Role of angiotensin II, endothelin-1, and nitric oxide in HgCl2-induced acute renal failure.

To elucidate the mechanisms underlying the development of HgCl2-induced acute renal failure (ARF), we examined the expression of endothelin (ET)-1, endothelial (e) nitric oxide synthase (NOS) and inducible (i) NOS, and a role of angiotensin II (ANG II) and tumor necrosis factor (TNF) in glomeruli and cortices from rats at 20 h after exposure of HgCl2. Prepro-ET-1 and iNOS mRNA were significantly increased in glomeruli and cortices from rats with HgCl2-induced ARF. However, eNOS mRNA was markedly decreased in glomeruli of rats with HgCl2-induced ARF. Blockade of the action of endogenous ANG II with TCV-116, an ANG II receptor type 1 antagonist, or prior administration of TNF antibody (Ab) neutralizing TNF bioactivity or aminoguanidine, an iNOS inhibitor, substantially suppressed the increase in the expression of prepro-ET-1 or iNOS mRNA seen in rats with HgCl2-induced ARF. Both TCV-116 and TNF Ab had no effects on the expression of eNOS mRNA. The abundance of ET-1, iNOS, and eNOS proteins was paralleled by the magnitude of each mRNA expression. Additionally, the aggravation of blood urea nitrogen and serum Cr observed in rats with HgCl2-induced ARF were significantly ameliorated together with the alleviation of proximal tubule epithelial cell injury when the expression of prepro-ET-1 or iNOS mRNA was blunted by prior administration of TCV-116 or prior injection of TNF Ab or aminoguanidine. These observations indicate that ANG II, ET-1, and NO may play an important role in the progression of HgCl2-induced ARF through the acceleration of proximal tubule epithelial cell injury and the deterioration of glomerular hemodynamics. In HgCl2-induced ARF, the gene expression of ET-1 or iNOS is at least in part up-regulated at the transcription level by endogenous ANG II or TNF.

Intracellular aggregate formation of dentatorubral-pallidoluysian atrophy (DRPLA) protein with the extended polyglutamine.

Dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant neurodegenerative disorder caused by the abnormal CAG triplet-repeat expansion resulting in an elongated polyglutamine (polyQ) stretch. We have recently showed that the DRPLA protein is cleaved during apoptosis by caspase-3, one of the cysteine protease family members known to be activated during apoptosis. We report here the subcellular localization of the DRPLA protein by fusing the green fluorescent protein as a tag. The full length DRPLA protein is localized predominantly but not exclusively in the nucleus regardless of the length of the polyQ stretch. In contrast, an N-terminal-deleted fragment containing polyQ produced by the proteolytic cleavage with caspase-3 is found both in the nucleus and the cytoplasm. Moreover, the same fragment with the elongated polyQ showed aggregation when overexpressed. Some cells with aggregate formation showed apoptotic phenotype. These findings raise the possibility that the DRPLA protein processed by caspase-3 may lead to aggregation of the protein resulting in the development of neurodegeneration.

[HgCl2-induced acute renal failure and its pathophysiology].

Mercury chloride (HgCl2) has a potent nephrotoxic effect. Most of Hg2+ existing in plasma following HgCl2 exposure forms a complex with sulfhydryl-containing ligands such as albumin and glutathione (GSH). The Hg(2+)-GSH complex is filtered in the glomeruli of the kidney and degraded into Hg(2+)-cysteine in the proximal tubules by the combined action of gamma-glutamyl transpeptidase and dipeptidase present in the epithelial cells. The degradation product is then incorporated and accumulated into the proximal tubule epithelial cells. The accumulated Hg2+ in the epithelial cells finally causes acute tubular necrosis (ATN) by its cytotoxic effect. At present, it is believed that tubular obstruction resulting from ATN triggers the onset of HgCl2-induced acute renal failure (ARF). A progressive fall in glomerular filtration rate (GFR) contributes to the progression of HgCl2-induced ARF. The fall in GFR may be caused by an increment in afferent arteriole resistance (RA) and a decrement in the ultrafiltration coefficient (Kf) due to mesangial cell contraction. These changes in RA and Kf may be attributed to the increased action of the vasoconstrictors, angiotensin II and endothelin-1 and to the decreased action of the vasodilator, nitric oxide observed at the glomerulus level of HgCl2-induced ARF. Accordingly, the imbalance between these vasoactive substances appears to play an important role in the progression of HgCl2-induced ARF due to reducing GFR. Further studies, however, remain to elucidate the mechanisms involved.

The molecular interaction of Fas and FAP-1. A tripeptide blocker of human Fas interaction with FAP-1 promotes Fas-induced apoptosis.

Fas (APO-1/CD95), which is a member of the tumor necrosis factor receptor superfamily, is a cell surface receptor that induces apoptosis. A protein tyrosine phosphatase, Fas-associated phosphatase-1 (FAP-1), that was previously identified as a Fas binding protein interacts with the C-terminal 15 amino acids of the regulatory domain of the Fas receptor. To identify the minimal region of the Fas C-terminal necessary for binding to FAP-1, we employed an in vitro inhibition assay of Fas/FAP-1 binding using a series of synthetic peptides as well as a screen of random peptide libraries by the yeast two-hybrid system. The results showed that the C-terminal three amino acids (SLV) of human Fas were necessary and sufficient for its interaction with the third PDZ (GLGF) domain of FAP-1. Furthermore, the direct cytoplasmic microinjection of this tripeptide (Ac-SLV) resulted in the induction of Fas-mediated apoptosis in a colon cancer cell line that expresses both Fas and FAP-1. Since t(S/T)X(V/L/I) motifs in the C termini of several other receptors have been shown to interact with PDZ domain in signal transducing molecules, this may represent a general motif for protein-protein interactions with important biological functions.

Analysis of C5a receptor by monoclonal antibody.

We prepared a mouse monoclonal antibody (mAb), termed 4C8, to the human C5a receptor (C5aR, CD88) by fusing spleen cells from mice immunized with mouse Ltk- cells transfected with cDNA of human C5aR (Ltk-/C5aR) to the mouse myeloma cell line P3U1. This mAb belonging to the IgM kappa subclass, detected a 43 kDa band on cell lysates of Ltk-/C5aR by immunoblotting analysis. Flow cytometry revealed that 4C8 specifically bound to Ltk-/C5aR, suggesting that this antibody is specific for C5aR. Furthermore, 4C8 was found to partially block both intracellular Ca2+ increase in PMN stimulated by C5a and 125I-C5a binding to C5aR on PMN. When cross-linked by anti-mouse IgM, 4C8 completely inhibited the binding of C5a to C5aR on PMN and Ltk-/C5aR. Therefore, it seems likely that this mAb does not recognize the C5aR active site but sterically inhibits the binding of C5a to its receptor. Using this mAb, we detected a 50 kDa band of C5aR on cell lysates of PMN, monocytes and platelets by immunoblotting. C5aR was expressed on PMN and monocytes as determined by flow cytometry, whereas it was not demonstrated on the surface of platelets. Based on these results, this mAb should be useful for analysis of C5aR expression in various immunological conditions and inflammatory diseases.

Regional characterization of G-protein subunits in glomeruli, cortices and medullas of the rat kidney.

We examined the types of guanine nucleotide-binding regulatory (G) protein subunits in isolated glomeruli, cortices excluding glomeruli and medullas of rat kidneys using bacterial toxin-catalyzed adenosine 5'-diphosphate (ADP) ribosylation and specific immunoblots. ADP ribosylation catalyzed by cholera or pertussis toxin revealed the presence of stimulatory G (Gs) or inhibitory G (Gi) proteins in membranes of the 3 segments of the kidney. Immunoblots further demonstrated the existence of several G-protein subunits, two Gs-protein alpha-subunits (G alpha s: 45 and 52 kD), Gi-protein alpha 1, alpha 2 and alpha 3-subunits (G alpha i1, G alpha i2: 40-41 kD, G alpha i3: 40 kD), bacterial toxin-insensitive G-protein alpha q- and alpha 11-subunits (G alpha q/11: 42 kD) and G-protein beta-subunits (G beta: 35-36 kD), in membranes of the preparations. The predominant subspecies of G alpha s was a 52-kD protein in glomerular membranes and a 45-kD protein in membranes of cortices and medullas. All of the G-protein subunits examined, however, were not detected in cytosolic fractions of glomeruli, cortices and medullas. Thus, we conclude that detectable quantities of several G-protein subunits including the new G-protein subunit, G alpha q/11, are present in membranes of glomeruli, cortices not containing glomeruli and medullas from the rat kidney. Both the existence of G alpha i1 and/or G alpha i2 subunits in glomeruli and the presence of G alpha q/11 subunits in the 3 preparations are new evidence.(ABSTRACT TRUNCATED AT 250 WORDS)

Bellini's duct tumor associated with end stage renal disease: a case diagnosed by lectin immunohistochemistry.

We report a hemodialysis patient with an atypical renal neoplasm. The tumor cells were arranged in a two-cell pattern similar to that in the usual excretory duct systems. The histochemical staining pattern with some lectins and monoclonal antibody corresponded to the distal nephrons of normal kidney tissue. These findings enabled us to diagnose this patient as having so-called Bellini's duct tumor.

[Cor triatriatum with situs inversus totalis: a case report of an infant].

Cor triatriatum associated with situs inversus is rare. A 6-month-old male who had cor triatriatum with atrial septal defect (ASD) and situs inversus totalis was successfully operated. Urgent surgical intervention was performed because of congestive cardiac failure despite drug therapy. The abnormal septum in the left atrium was resected and ASD was closed. His postoperative course has been uneventful.

Polycythemia associated with primary systemic amyloidosis: elevated levels of hemopoietic factors and cytokines.

A 60-year-old man was diagnosed to have primary systemic amyloidosis (AL type) and polycythemia (Hgb 19.1 g/dl, WBC 14,600/microliters, and platelets 60.5 x 10(4)/microliters). In vitro colony assay for hemopoietic stem cells showed increased numbers of colony-forming units (CFU)-G, CFU-GM, CFU-E, and CFU-Meg. Serum levels of erythropoietin (Epo), granulocyte colony-stimulating factor (G-CSF), interleukin (IL)-3 and IL-6 were elevated. The infiltration of amyloid fibrils into the hemopoietic factor-producing tissues may stimulate abnormal secretion of hemopoietic factors and cytokines, resulting in increased numbers of hemopoietic stem cells and polycythemia in the peripheral blood.

Protein intake affects levels of G-protein subunits G alpha i2, G alpha i3, and G beta in rat glomerular membranes.

Using toxin-catalyzed ADP-ribosylation and specific immunoblots we examined whether the mass of G-protein subunits, G alpha s, G alpha i (includes G alpha i2, G alpha i3, and G alpha 0) and G alpha beta, in glomerular membranes was altered by dietary protein intake. ADP-ribosylation catalyzed by cholera toxin (CT) or pertussis toxin (PT) detected significant amounts of G alpha s or G alpha i in glomerular membranes from rats fed a low (6% casein) or a high (40% casein) protein diet. There was no significant difference in G alpha s content between glomerular membranes from low or high protein-fed rats. However, the amounts of G alpha i were significantly lower in glomerular membranes from rats fed a high protein diet when compared to glomerular membranes from rats fed a low protein diet. Two isoforms of immunoreactive G alpha s, 45 and 52 kDa proteins, were detected in glomerular membranes. The predominant isoform of G alpha s was a 52 kDa protein. As with ADP-ribosylation, immunoblots showed no significant difference in G alpha s content between glomerular membranes obtained from the two diet groups of rats. Also, immunoreactive G alpha i2, G alpha i3 and G beta were present in glomerular membranes. The mass of G alpha i2 and G alpha i3 was significantly lower in glomerular membranes of rats fed a high protein diet than in those of rats fed a low-protein diet. The decreased mass of total G alpha i, that is G alpha i2 and G alpha i3, was comparable to that seen with PT-catalyzed ADP-ribosylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Bilateral ureteral obstruction alters levels of the G-protein subunits G alpha s and G alpha q/11.

To evaluate the effects of bilateral ureteral obstruction (BUO) on the levels of G-protein subunits in glomeruli, we examined the types and amounts of G-protein subunits in glomerular membranes from sham-operated control (SOC) rats and rats with BUO of 24 hours duration utilizing bacterial toxin-catalyzed ADP-ribosylation and specific antibodies. ADP-ribosylation catalyzed by cholera or pertussis toxin demonstrated the presence of Gs and Gi proteins in glomerular membranes. Immunoblots further revealed the existence of two types of G alpha s (45 and 52 kDa), as well as G alpha i2 (40 kDa), G alpha i3 (41 kDa), G alpha q/11 (42 kDa) and G beta (35 to 36 kDa) in glomerular membranes. The predominant subspecies of G alpha s was the 52 kDa protein. Detectable amounts of G alpha o were not found in glomerular membranes. Moreover, G-protein subunits were not detected in cytosolic extracts of glomeruli. Both forms of G alpha s and G alpha q/11 were significantly reduced in glomerular membranes from rats with BUO when compared to SOC rats. No significant difference in total G alpha i, G alpha i2 and G alpha i3 and G beta content was observed between the two groups of rats. In vivo pretreatment of rats with simultaneous administration of the angiotensin-converting enzyme inhibitor, enalaprilat, and the thromboxane synthase inhibitor, OKY-046, maintained the amount of G alpha s and G alpha q/11 in rats with BUO at the levels seen in SOC rats. The two drugs did not affect the amounts of G-protein subunits in glomerular membranes of SOC rats.(ABSTRACT TRUNCATED AT 250 WORDS)