Lipopolysaccharide alters ecto-ATP-diphosphohydrolase and causes relocation of its reaction product in experimental intrahepatic cholestasis

Lipopolysaccharide (LPS), a bacterial endotoxin, exerts profound inflammatory actions toward various tissues and cells. We induced intrahepatic cholestasis in rats by administration of LPS and followed ecto-ATP-diphosphohydrolase (ecto-apyrase) activity in the liver. The activity of the enzyme had decreased to 77% 2 h after injection compared with the activity in control animals. The maximum decrease was detected 24 h after administration. The activity was found to have partially recovered 1 week after injection, but had yet to reach control levels. In contrast to the decrease in ecto-apyrase activity, there were increases in alkaline phosphatase activity and bilirubin concentration, markers of cholestasis. In response to LPS, the reaction product of ecto-apyrase was found to relocate from the canalicular domain of the plasma membrane of hepatocytes, its predominant localization in the liver of intact animals, to the basolateral and sinusoidal domains. The pattern of histochemical reaction indicated modulation of the enzyme activity and changes in trafficking of intracellular proteins. Taken together, our findings showed that LPS administration alters ecto-apyrase and causes relocation of its reaction product from the canalicular domain of the plasma membrane of hepatocytes in the rat. It is suggested that relocation of the reaction product may be a protective mechanism to enable the hepatocytes to withstand the cytokine-induced metabolic perturbations.     

A simple approach for the analysis of intracellular movement of oxidant-producing intracellular compartments in living human neutrophils

In human neutrophils, superoxide is generated primarily within specialized oxidant-producing intracellular compartments. The present study employs a simple methodological approach to evaluate the intracellular movement of these structures in living human neutrophils. Using a CCD camera system, we monitored fluorescence in cells loaded with the succinimidyl ester of dichlorodihydrofluorescein diacetate, which is nonfluorescent until oxidized by reactive oxygen species. Fluorescence-positive intracellular compartments became detectable after neutrophils were stimulated with phorbol myristate acetate for 1 min. Further stimulation increased the intracellular compartments in both number and size in a time-dependent manner. Upon stimulation with phorbol myristate acetate, no fluorescence was seen in intracellular compartments of neutrophils isolated from patients with X-linked chronic granulomatous disease lacking gp91-phox, a membrane component of NADPH oxidase. The method enables tracking of the movement of a single oxidant-producing intracellular compartment following cell stimulation and visualization of the intracellular structures formed by fusion of oxidant-producing intracellular compartments with endocytotic vesicles and phagosomes. Therefore, it is considered to be an informative tool for evaluation of the intracellular dynamics of oxidant-producing intracellular compartments in living human neutrophils and may have a diagnostic value.     

Novel mannose-binding rice lectin composed of some isolectins and its relation to a stress-inducible salT gene

The novel mannose-binding rice lectin (MRL) purified by Sephadex G-50 or maltamyl Sepharose 4B affinity chromatography was not homogeneous, but the components were separated clearly by two dimensional polyacrylamide gel electrophoresis (1st; isoelectric focusing with Immobiline, 2nd; SDS-PAGE). The major spots were located at pI 4.85 and 4.74, and minor spots at pI 4.66, 4.56, and 4.44; all spots were distributed at about MW 45,000. Other faint spots were sometimes detected just below the major spots. In the western blot analysis, all the spots reacted with the monoclonal antibodies specific to MRL, which bound to MRL and inhibited the lectin activity to agglutinate rabbit erythrocytes. The proteins of the spots at pI 4.85, 4.77, 4.66, and 4.56 had lectin activity. The major proteins at pI 4.85 and 4.77 also had the common amino acid sequence at N-terminus, TLVKIGPWGGNGGSAQDISV, which is almost identical to salt and drought stress-inducible salT gene products in rice plants. High homology was also conserved in both the cDNA and the genomic clones encoding the MRL component at pI 4.85, which were selected with MRL-specific antibodies and an oligonucleotide designed from the partial amino acid sequence. All results suggest that MRL is composed of several isolectins, if not, related proteins having a common epitope and may belong to a family of stress-inducible proteins.     

Ataxia-telangiectasia: identification and detection of founder-effect mutations in the ATM gene in ethnic populations.

To facilitate the evaluation of ATM heterozygotes for susceptibility to other diseases, such as breast cancer, we have attempted to define the most common mutations and their frequencies in ataxia-telangiectasia (A-T) homozygotes from 10 ethnic populations. Both genomic mutations and their effects on cDNA were characterized. Protein-truncation testing of the entire ATM cDNA detected 92 (66%) truncating mutations in 140 mutant alleles screened. The haplotyping of patients with identical mutations indicates that almost all of these represent common ancestry and that very few spontaneously recurring ATM mutations exist. Assays requiring minimal amounts of genomic DNA were designed to allow rapid screening for common ethnic mutations. These rapid assays detected mutations in 76% of Costa Rican patients (3), 50% of Norwegian patients (1), 25% of Polish patients (4), and 14% of Italian patients (1), as well as in patients of Amish/Mennonite and Irish English backgrounds. Additional mutations were observed in Japanese, Utah Mormon, and African American patients. These assays should facilitate screening for A-T heterozygotes in the populations studied.     

Proteasome inhibition suppresses DNA-dependent protein kinase activation caused by camptothecin

The ubiquitin–proteasome pathway plays an important role in DNA damage signaling and repair by facilitating the recruitment and activation of DNA repair factors and signaling proteins at sites of damaged chromatin. Proteasome activity is generally not thought to be required for activation of apical signaling kinases including the PI3K-related kinases (PIKKs) ATM, ATR, and DNA-PK that orchestrate downstream signaling cascades in response to diverse genotoxic stimuli. In a previous work, we showed that inhibition of the proteasome by MG-132 suppressed 53BP1 (p53 binding protein1) phosphorylation as well as RPA2 (replication protein A2) phosphorylation in response to the topoisomerase I (TopI) poison camptothecin (CPT). To address the mechanism of proteasome-dependent RPA2 phosphorylation, we investigated the effects of proteasome inhibitors on the upstream PIKKs. MG-132 sharply suppressed CPT-induced DNA-PKcs autophosphorylation, a marker of the activation, whereas the phosphorylation of ATM and ATR substrates was only slightly suppressed by MG-132, suggesting that DNA-PK among the PIKKs is specifically regulated by the proteasome in response to CPT. On the other hand, MG-132 did not suppress DNA-PK activation in response to UV or IR. MG-132 blocked the interaction between DNA-PKcs and Ku heterodimer enhanced by CPT, and hydroxyurea pre-treatment completely abolished CPT-induced DNA-PKcs autophosphorylation, indicating a requirement for ongoing DNA replication. CPT-induced TopI degradation occurred independent of DNA-PK activation, suggesting that DNA-PK activation does not require degradation of trapped TopI complexes. The combined results suggest that CPT-dependent replication fork collapse activates DNA-PK signaling through a proteasome dependent, TopI degradation-independent pathway. The implications of DNA-PK activation in the context of TopI poison-based therapies are discussed.     

DNA Lesions Induced by Replication Stress Trigger Mitotic Aberration and Tetraploidy Development

During tumorigenesis, cells acquire immortality in association with the development of genomic instability. However, it is still elusive how genomic instability spontaneously generates during the process of tumorigenesis. Here, we show that precancerous DNA lesions induced by oncogene acceleration, which induce situations identical to the initial stages of cancer development, trigger tetraploidy/aneuploidy generation in association with mitotic aberration. Although oncogene acceleration primarily induces DNA replication stress and the resulting lesions in the S phase, these lesions are carried over into the M phase and cause cytokinesis failure and genomic instability. Unlike directly induced DNA double-strand breaks, DNA replication stress-associated lesions are cryptogenic and pass through cell-cycle checkpoints due to limited and ineffective activation of checkpoint factors. Furthermore, since damaged M-phase cells still progress in mitotic steps, these cells result in chromosomal mis-segregation, cytokinesis failure and the resulting tetraploidy generation. Thus, our results reveal a process of genomic instability generation triggered by precancerous DNA replication stress.     

Clinical application of Clostridium botulinum type A neurotoxin purified by a simple procedure for patients with urinary incontinence caused by refractory destrusor overactivity

Type A neurotoxin of Clostridium botulinum was purified by a simple procedure using a lactose gel column. This procedure was previously reported for type B neurotoxin. Hemagglutinin-positive toxins (19S and 16S) were bound to the column under acid conditions, and the neurotoxin alone was dissociated from these hemagglutinin-positive toxins by changing the pH of the column to an alkaline condition. The toxicity of this purified toxin preparation was retained for at least 1 year at -30 degrees C by supplementing it with either 0.1% albumin or 0.05% albumin plus 1% trehalose. This preparation was used to treat 18 patients with urinary incontinence caused by refractory idiopathic and neurogenic detrusor overactivity; 16 of the patients showed excellent improvement. Improvements started within 1 week after injection in most cases and lasted 3-12 months [corrected]     

Repellent from traditional Chinese medicine, Periploca sepium Bunge

By using a new bioassay-guided method, 2-hydroxy-4-methoxybenzaldehyde isolated from the root bark of Periploca sepium, a traditional Chinese medicine, showed repellent activity against the olive weevil (Dyscerus perforatus) at 62.5, 125, 250 and 500 microg/disc, respectively. In addition, it also exhibited antinematodal activity against Bursaphelenchus xylophilus at a minimum effective dose of 200 microg/ball. The three related compounds obtained were also evaluated for the above-mentioned bioactivities.